We examined the roles of Ca2+ and protein kinase C (PKC) in the cilio-excitatory response to serotonin in pedal ciliary cells from Helisoma trivolvis embryos. Serotonin (5-hydroxytryptamine; 5-HT; 100 micromol/L) induced an increase in ciliary beat frequency (CBF) was abolished by microinjected BAPTA (50 mmol/L), but was only partially inhibited by the phospholipase C inhibitor U-73122 (10 micromol/L). The diacylglycerol analogs 1-oleoyl-2-acetyl-sn-glycerol (100 micromol/L) and 1,2-dioctanoyl-sn-glycerol (100 micromol/L) caused increases in [Ca2+]i that were smaller than those induced by serotonin. In the absence of extracellular Ca2+, 1,2-dioctanoyl-sn-glycerol (100 micromol/L) failed to elicit an increase in both CBF and [Ca2+]i. In contrast, the serotonin-induced increase in CBF persisted in the absence of extracellular Ca2+, although the increase in [Ca2+]i was abolished. PKC inhibitors bisindolylmaleimide (10 and 100 nmol/L) and calphostin C (10 nmol/L) partially inhibited the serotonin-induced increase in CBF, but didn't affect the serotonin-induced change in [Ca2+]i. These findings suggest that an intracellular store-dependent increase in [Ca2+]i mediates the cilio-excitatory response to serotonin. Furthermore, although PKC is able to cause an increase in [Ca2+]i through calcium influx, it contributes to the cilio-excitatory response to 5-HT through a different mechanism.