The effects of delta8- and delta9-tetrahydrocannabinol on the biosynthesis of 3H-acetylcholine (ACh) from 3H-choline in cortical, hypothalamic and striatal rat brain slices were examined. The two cannabinols were found to inhibit the synthesis of 3H-ACh in the three brain regions. Treatment with cannabidiol did not alter ACh synthesis. Delta8-tetrahydrocannabinol was approximately twice as effective as the delta9-isomer. This effect was not associated with alterations in striatal and cortical choline acetyltransferase or with an impaired high-affinity uptake system for choline in the striatum. Treatment with delta8- and delta9-cannabinols, likewise, did not change striatal choline and ACh levels. Antagonism of the ACh biosynthesis inhibition occurred when slices from treated animals were incubated in depolarizing concentration of K+ ion. These results suggest that the inhibition of ACh synthesis observed in tetrahydrocannabinol-treated rats may be related to interference with the propagated action potential or with the depolarization process in cholinergic neurons.