The larval brain of the tobacco hornworm, Manduca sexta, was maintained in vitro and the uptake of labelled and unlabelled choline as well as their subsequent metabolism were measured by high-voltage paper electrophoresis. Significant levels of choline lipid metabolites, phosphorylcholine and acetylcholine (ACh) were noted. Unbound choline reached equilibrium after 6-8 h of incubation, while ACh accumulation continued to increase after 24 h indicating that the rate of synthesis exceeded the rate of breakdown. An apparent Km could not be determined for these whole-organ studies; however, the Vmax for ACh accumulation for days 5 (70 pmol/brain/h) and 6 (105 pmol/brain/h) of the last larval instar did vary significantly while the level of unbound choline in the brain did not change. The level of choline uptake was dependent upon the presence of Na+ and Ca2+, while the amount of ACh accumulated was affected specifically by the presence of Mg2+, the latter ion activating acetylcholinesterase. The determination of levels of unbound choline and ACh accumulation in the developing brain during the last two larval instars demonstrated increases in acetylcholine accumulation at previously reported times of the release of the hormone that initiates the molting process, prothoracicotropic hormone. These changes in the patterns of ACh accumulation occur during 4-8 h time intervals; this is the first report of such short-range changes in neurotransmitter metabolism in whole brains. The intensity of the ACh accumulation shift is equivalent to the intensity of the hormone burst. Other fluctuations in the levels of ACh accumulation and free choline correlate with the development of the brain.