Developmental changes in the synthesis of acetylcholine (ACh) were investigated in rat hippocampus and frontal cortex. Particular reference was made to the conversion, into ACh, of the choline accumulated by high-affinity uptake as defined using 1 microM hemicholinium-3 (HC-3). Using solutions containing 11.1 mM glucose, conversions were respectively 31 and 55%, in fine slices from 4-8-day-olds. Free choline accounted very largely for the remainder of the choline accumulated. In samples from adults, ACh accounted for 80% of the uptake. The inefficient conversions (into ACh) in immature brain were not the result of a requirement for ketone bodies as the source of acetyl-coenzyme A (acetyl-CoA). Greater rates of release of newly synthesised ACh, than in mature samples, were not responsible, neither were greater cholinesterase activities. The stimulation of high-affinity choline uptake, caused by prior depolarisation of the tissues using K+, also increased during development from 78 to 238% with hippocampus and from 49 to 170% with frontal cortex. Furthermore, prior depolarisation increased the efficiency with which choline, accumulated by high-affinity uptake, was converted into ACh. At all stages of development 80% of the additional choline accumulated, after depolarisation, was converted into ACh. It is concluded that the specificity of HC-3-sensitive uptake is incomplete in immature brain, i.e. high-affinity choline uptake is not exclusively into cholinergic neurones. The cholinergic neuronal compartment becomes more prominent during development so that the specificity is complete in mature brain.(ABSTRACT TRUNCATED AT 250 WORDS)