1. The development of the adenosine A1 and A2b receptors inducing relaxation of the rat duodenum was studied by use of a combination of functional and radioligand binding assays on rats aged between 5 and 30 days and compared with results previously found in adult rat duodenum. 2. 1,3-[3H]-dipropyl-8-cyclopentylxanthine ([3H]-DPCPX) bound with high affinity to a single site in duodenum preparations from rats aged 20, 25 and 30 days. At 10 and 15 days there was no detectable specific binding of [3H]-DPCPX. 3. The affinity (KD) of the binding site for [3H]-DPCPX was similar in membrane preparations from 20, 25 and 30 day old animals (1.58-2.27 nM), but the density (Bmax) of binding sites was found to increase up to 25 days where peak levels (72.0 +/- 9.5 fmol mg-1 protein) were observed and then decline at 30 days (45.5 +/- 2.9 fmol mg-1 protein) to levels commensurate with those previously determined in the adult rat duodenum. 4. In duodenum from 10 day old rats no responses to N6-cyclopentyladenosine (CPA, 1 nM-10 microM) were observed, at 15 days the duodenum responded to the highest concentration of CPA (3 microM) only, and at 20-30 days concentration-related responses were observed, with the potency of CPA increasing with an increase in age. DPCPX (10 nM) abolished the responses to CPA except at the highest concentration of CPA (3 microM) where the response was markedly attenuated, suggesting the presence of an A1, receptor. 5. In rat duodenum from animals of all ages (5-30 days), concentration-related responses to 5'-N-ethylcarboxamidoadenosine (NECA) were observed. The potency of NECA remained constant with an increase in age, whereas the maximum relaxation response increased from 20% at 5 days to 110% at 25 and 30 days. In the presence of 1 microM DPCPX a right-ward shift in the concentration-response curve to NECA was observed at all ages. In the presence of 10 nM DPCPX, the response to NECA was unaffected in the duodenum from animals aged 10 and 15 days. However, in duodenum from animals aged 20-30 days the concentration-response curve to NECA was shifted to the right suggesting that there is an A1 component to the action of NECA at these ages. Schild analysis of the effects of increasing concentrations of DPCPX versus NECA on the duodenum from 25 day old animals generated a slope of 0.62 suggesting that NECA acts at A1 and A2b receptors as in the adult. 6. The A2b-selective analogue, 2-[p-(carboxyethyl)-phenylethylamino]-5'-N-ethylcarboxamidoadenosi ne (CGS 21680) (10 nM-10 microM) was without effect on the carbachol-contracted duodenum from 15 day old rats and the duodenum from 25 day old rats responded to the highest concentration of CGS 21680 only, suggesting that the A2 receptors here, as in the adult, are not of the A2a subtype. The adenosine antagonist, 8-phenyltheophylline (8-PT) (10 microM), abolished the inhibitory effects of NECA (100 nM-100 microM) on 10, 15 and 25 day old rat duodenum indicating that the responses to NECA were not mediated via an adenosine A3 receptor. 7. These results show that adenosine A1 receptors in rat duodenum are present and functionally viable from day 20 onwards and that the density of A1 receptors varies with age, increasing up to day 25 and then declining at day 30 to a density commensurate with that found in the adult. The responses to CPA, mediated via the A1 receptor, increase with age in a similar fashion. In contrast however, the response to NECA was evident from day 5, the earliest age studied, and from days 5-15 NECA acted via the A2b receptor subtype. However, from day 20 onwards NECA acted at a mixed population of A1 and A2b receptors. These results demonstrate the differential development of the A1 and the A2b receptors in the rat duodenum.