Sarafotoxin b (S6b)-induced changes in intracellular Ca2+ concentration ([Ca2+]i) were monitored in cultured canine tracheal smooth muscle cells (TSMCs) by a fluorescent Ca2+ indicator fura-2. S6b elicited an initial transient peak followed by a sustained elevation of [Ca2+]i. BQ-123, an endothelin-A (ETA) receptor antagonist, had a high affinity to block the rise in [Ca2+]i response to S6b. In the absence of external Ca2+, only an initial transient peak of [Ca2+]i was seen, the sustained elevation of [Ca2+]i could then be evoked by addition of 1.8 mM Ca2+. Ca2+ influx was required for the changes of [Ca2+]i, since the Ca(2+)-channel blockers, diltiazem, verapamil, and Ni2+, decreased both the initial and sustained elevation of [Ca2+]i in response to S6b. TSMCs pretreated with phorbol 12-myristate 13-acetate (PMA, 1 microM) for 30 min attenuated Ca2+ mobilization induced by S6b, which was reversed by staurosporine, a protein kinase C (PKC) inhibitor. The change of [Ca2+]i induced by S6b was attenuated by cholera toxin pretreatment, but not by pertussis toxin. These data demonstrate that the initial detectable increase in [Ca2+]i stimulated by S6b is due to the activation of ETA receptors and subsequent release of Ca2+ from internal stores, whereas the contribution of external Ca2+ follows and partially involves a diltiazem- and verapamil-sensitive process. The inhibition of PMA on S6b-induced Ca2+ mobilization was inversely correlated with membraneous PKC activity.