The effects of 2-(4-phenylpiperidino)cyclohexanol (AH5183) and quinacrine, two potent inhibitors of acetylcholine transport into vesicles isolated from Torpedo electric organ, were examined on acetylcholine metabolism in rat cortical slices. K+-stimulated acetylcholine release was reduced in a concentration-dependent manner by AH5183 and quinacrine, with IC50 values of 1 microM and 50 microM, respectively. Both drugs also reduced the synthesis of acetylcholine in slices and inhibited synaptosomal high affinity choline transport. The inhibitory effect of AH5183 appears to be directed primarily on the release of acetylcholine while the major effect of quinacrine is on the synthesis of acetylcholine. Examination of the subcellular distribution of acetylcholine in brain slices incubated in high K+ showed that AH5183 increased S3 (cytoplasmic) acetylcholine levels but did not alter P3 (vesicular) acetylcholine levels. P3 acetylcholine levels were reduced by AH5183 in a low K+ media while the S3 acetylcholine levels were the same as controls. These results are consistent with the concept that there is a small, active, highly labile fraction of vesicles that are the source of the released acetylcholine and that the loading of these vesicles is blocked by AH5183.