Abstract: The biochemical mechanisms involved in the regulation of acetylcholine (ACh) turnover are poorly understood. In the experiments reported here, we examined whether inhibition of the serine/threonine phosphatases 1 and 2A by calyculin A or okadaic acid alters ACh synthesis by rat hippocampal preparations. With hippocampal slices, calyculin A (50 nM) and okadaic acid (50 nM) reduced significantly (p < 0.01) the synthesis of [3H]ACh from [3H]choline. Both calyculin A and okadaic acid produced significant depletion of endogenous tissue ACh in a concentration-dependent manner (p < 0.01). This depletion was not the result of a drug-induced increase of spontaneous ACh release, which was not changed significantly (p > 0.7) by either drug. Choline acetyltransferase (ChAT) activity from tissue exposed to calyculin A or okadaic acid was reduced in a concentration-dependent manner (p < 0.05), but these phosphatase inhibitors did not act directly on ChAT in vitro; i.e., enzymatic activity was not altered significantly (p > 0.4) in the presence of calyculin A or okadaic acid. Both high-affinity and low-affinity [3H]choline uptake by hippocampal synaptosomes were reduced significantly in a concentration-dependent manner in the presence of calyculin A or okadaic acid; these agents reduced Vmax values for high- and low-affinity choline uptake (p < 0.01) with no significant change in Km values (p > 0.1), indicating a noncompetitive inhibition. Taken together, these data suggest that phosphatase activity plays a role in presynaptic central cholinergic nerve terminal function, in particular in the modulation of ACh synthesis.