The cholinergic vesicular uptake blocker, 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid (AH5183), had several effects on presynaptic cholinergic function that depended on the duration of treatment and dose. The synthesis, storage and release of newly synthesized [3H]ACh were monitored because the vesicular uptake of this pool of transmitter may be preferentially affected by the drug. Initially, high concentrations of AH5183 (over 10 microM) increased the spontaneous release but decreased the K+ depolarization-induced release of newly synthesized transmitter. [3H]Choline efflux was not altered by the drug. High affinity choline uptake was slightly (10-20%) inhibited by AH5183 in an apparently competitive but time-dependent manner. In contrast to its initial effects on [3H]ACh release, AH5183 (50nM-100 microM) very potently inhibited both the spontaneous and K+-induced release of [3H]ACh but not of [3H]choline after a 60 min preincubation. [3H]ACh levels in cytoplasmic (S3) and crude membrane (P3) fractions were not affected by a 2-min incubation with 10 microM AH5183. After a 60-min preincubation with this drug dose, however, the P3 and S3 levels of newly synthesized transmitter were decreased and increased, respectively. Subsequent fractionation of synaptosomes by sucrose-density gradient centrifugation revealed that these reductions in P3 [3H]ACh-levels were referable to reductions in two subfractions D and H that have been reported to contain low density vesicles and denser vesicles associated with plasma membranes, respectively.