We examined, using physiological and morphological techniques, the distribution of Ca(2+)-gated K+ (gKca) channels relative to the location of Ca2+ channels and transmitter release sites at the frog neuromuscular junction (NM). Charybdotoxin (ChTx) and iberiotoxin, blockers of gKca channels with large conductances, increase transmitter release at the frog NMJ. Intracellular Ca2+ buffers with rapid binding kinetics, dimethyl BAPTA and BAPTA, prevented the effect of ChTx, but EGTA, a Ca2+ buffer with similar affinity for Ca2+ but slower binding kinetics, did not. Dimethyl BAPTA and BAPTA, but not EGTA, caused a temporary increase in transmitter release. Labeling of gKca channels with ChTx-biotin revealed a series of bands located at the sites of Ca2+ channels, but this labeling did not occur in denervated preparations. Cross sections of NMJs revealed that gKca channels are clustered in the presynaptic membrane facing the postsynaptic membrane. We conclude that gKca channels are strategically clustered at the neurotransmitter release sites, where they can be quickly activated by Ca2+ entering the terminal.