Abstract Motility of trout sperm is suppressed by extracellular K+ at rest and initiated by the decrease in the extracellular K+ concentration, and Ca2+ and cyclic AMP participate in the cell signaling for the initiation of sperm motility. We showed here that tetraethyl ammonium (TEA) and some blockers used as the voltage-dependent K+ channel blockers in the neuron inhibited the motility in the sperm of the rainbow trout and the steelhead trout, Oncorhynchus mykiss. Other types of K+ channel blockers, e.g., apamin, a blocker of small conductance Ca2+-dependent K+ channels, did not inhibit sperm motility. L-type Ca2+ channel blockers such as nifedipine, nimodipine, etc. also inhibited the motility, but other types of Ca2+ channel blockers did not. On the other hand, the gradual increase in external K+ concentration caused both gradual decreases in the amplitude of hyperpolarization of the sperm plasma membrane and cyclic AMP synthesis. TEA and nifedipin suppressed both hyperpolarization and cyclic AMP synthesis, and these suppressions were relieved by addition of the K+ ionophore, valinomycin. The calmodulin inhibitors W-7, trifluoroperazine and calmidazol-Cl inhibited the sperm motility, membrane hyperpolarization and cyclic AMP synthesis, although only at rather high concentrations. These results suggest that K+ efflux through K+ channels and Ca2+ influx through Ca2+ channels that are sensitive to specific channel blockers cause the changes of membrane potential and lead to a synthesis of cyclic AMP in the cell signaling for the initiation of trout sperm motility.