1. In an attempt to explain the previous electrophysiological data on the ontogeny of beta-adrenergic and muscarinic cholinergic interactions on cardiac Ca2+ current, biochemical studies were performed on the ontogeny of beta-adrenoceptors, muscarinic cholinoceptors and Ca2+ channels in cardiac muscle of developing rats: 16-20 days old foetuses, 0-20 days old neonates, and 2-3 months old adults. 2. Developmental changes in cardiac beta-adrenoceptors, muscarinic cholinoceptors, and Ca2+ channels were determined with the use of specific radioligands, [3H]-dihydroalprenolol (DNA), [3H]-quinuclidinyl benzilate (QNB), and [3H]-nitrendipine (NTD), respectively. 3. The Bmax value (fmol mg-1 tissue) for [3H]-DNA binding started to increase on post-gestation day 20, reached almost its maximum level on neonatal day 6, kept almost the same level until neonatal day 20, and then decreased slightly to its adult level. 4. The Bmax value (fmol mg-1 tissue) for [3H]-QNB binding started to increase on post-gestation day 16, reached almost its maximum level on neonatal day 0, remained almost constant until neonatal day 15, and then decreased to its adult level. 5. The Bmax value (fmol mg-1 tissue) for [3H]-NTD binding increased with age between post-gestation day 18 and neonatal day 15, stayed almost constant until neonatal day 20, and then decreased to its adult level. 6. The Kd values for [3H]-DHA, [3H]-QNB, and [3H]-NTD bindings remained almost constant during the developmental period examined. 7. Isoprenaline (Iso) increased the kx of slow action potentials (APs) from post-gestation day 18, and the adult level was reached at about 2 weeks after birth; this developmental time course is similar to that of Ca2+ channels. The number of beta-adrenoceptors also started to increase a few days before birth, but attained its peak about one week earlier than did the Pax of slow APs or the number of Ca2 + channels. 8. Acetylcholine (ACh) almost completely abolished the Iso-induced increase in m,,ax observed from postgestation day 18 to neonatal day 20; this developmental time course for the ACh effect is consistent with the finding that the number of muscarinic cholinoceptors started to increase on post-gestation day 16 and reached a peak on the day of birth. 9. Previous electrophysiological and the present biochemical findings strongly suggest that the functional coupling between muscarinic cholinoceptors and Ca2+ current is already established when the coupling between beta-adrenoceptors and Ca2 + current starts to operate in developing rat hearts.