The primary target for cocaine is believed to be monoamine transporters because of cocaine's high-affinity binding that prevents re-uptake of released neurotransmitter. However, direct interaction with ion channels has been shown to be important for certain pharmacological/toxicological effects of cocaine. Here I show that cocaine selectively blocks a calcium-dependent K(+) channel in hippocampal neurons grown in culture (IC(50)=approximately 30 microM). Single-channel recordings show that in the presence of cocaine, the channel openings are interrupted with brief closures (flicker block). As the concentration of cocaine is increased the open-time is reduced, whereas the duration of brief closures is independent of concentration. The association and dissociation rate constants of cocaine for the neuronal Ca(2+)-activated K(+ )channels are 261+/-37 microM: (-1)s(-1) and 11451+/-1467 s(-1). The equilibrium dissociation constant (K(B)) for cocaine, determined from single-channel parameters, is 43 microM. The lack of voltage dependence of block suggests that cocaine probably binds to a site at the mouth of the pore. Block of Ca(2+)-dependent K(+) channels by cocaine may be involved in functions that include broadening of the action potential, which would facilitate transmitter release, enhancement of smooth muscle contraction particularly in blood vessels, and modulation of repetitive neuronal firing by altering the repolarization and afterhyperpolarization phases of the action potential.