We have examined the question of how regenerating sympathetic neurons that are concomitantly induced to become cholinergic regulate choline allocation between ACh and the phospholipid synthetic pathway. The allocation of choline into ACh increased parabolically with time in culture, and by 3 weeks, cultures with neurites of approximately 6 mm length were incorporating over 85% of the choline locally in the neurites into four major metabolites: ACh, phosphorylcholine, cytidine diphosphocholine, and phosphatidylcholine. The near-equivalent distribution of labeled choline between intracellular choline, ACh, and phosphorylcholine was independent of time (5 min to 6 hr) and choline concentration (0.125–30 microM), phosphatidylcholine being the sole metabolite whose level in the neurites increased steadily with incubation time. Relative choline distribution into ACh and phosphorylcholine was unaltered even after a brief depolarizing prepulse, which caused a two- to fourfold enhancement in the total choline incorporated. These observations, allied with the similar half- saturation constants and Vmax values of CAT and choline kinase for intracellular choline, suggest that growing sympathetic neurons are poised to allocate choline symmetrically between the synthesis of ACh and phosphatidylcholine in the neurites. When, however, the supply of choline was limited either by replacement of Na+ in the medium with N- methyl-D-glucamine, or by vesamicol, a 90–97% reduction in intracellular choline caused a similar decline in ACh levels but synthesis of metabolites of the phosphatidylcholine pathway was maintained unperturbed, as if no drug was present. We suggest that this can be accounted for by a 10-fold increase in choline kinase activity. Thus, growing sympathetic neurons that express cholinergic properties not only maintain their chief cellular phosphatidylcholine-synthesizing activity concomitantly with ACh synthesis in the neurites, but may also preserve phosphatidylcholine synthesis more effectively than ACh synthesis when the supply of choline is perturbed. Relinquishing ACh synthesis during growth may be one way of conserving and encouraging neurite regeneration.