The purpose of the present study was to investigate the role and type of Ca 2+ channels involved in the stimulatory effects of endothelin-1 (ET-1) on the Ca 2+-dependent functional responses, p42/p44 MAP kinase phosphorylation, 20-kDa myosin light chain (MLC) phosphorylation and contraction, in rabbit iris sphincter, a nonvascular smooth muscle. ET-1 induced inositol phosphates production, MAP kinase phosphorylation, MLC phosphorylation (MLC 20-P plus MLC 20-2P) and contraction in a concentration-dependent manner with EC 50 values of 71, 8, 6 and 25 nM, respectively. ET-1-induced MAP kinase phosphorylation, MLC phosphorylation and contraction were not significantly affected by nifedipine (1–60 μM), an L-type Ca 2+ channel blocker, or by LOE 908 (1–100 μM), a blocker of Ca 2+-permeable nonselective cation channels. However, SKF96365, a receptor-operated Ca 2+ channel (ROCC) blocker, inhibited MAP kinase phosphorylation, MLC phosphorylation and contraction in a concentration-dependent manner with IC 50 values of 28, 30 and 42 μM, respectively. 2-APB, a store-operated Ca 2+ channel (SOCC) blocker, inhibited ET-1-induced MLC phosphorylation and contraction in a concentration-dependent manner with IC 50 values of 12.7 and 19 μM, respectively, but was without effect on MAP kinase phosphorylation. The combined effects of submaximal concentrations of SKF96365 and 2-APB on ET-1-induced MLC phosphorylation and contraction were not additive, implying that their inhibitory actions could be mediated through a common Ca 2+ entry channel. PD98059, a MAP kinase inhibitor, had no effect on ET-1-induced MLC phosphorylation and contraction, suggesting that these ET-1 effects in the rabbit iris muscle are MAP kinase-independent. In conclusion, the present study demonstrated for the first time that in rabbit iris sphincter (a) ET-1, through the ET A receptor, stimulates MAP kinase phosphorylation, MLC phosphorylation and contraction in a concentration-dependent manner, (b) that these Ca 2+-dependent functional responses are not significantly affected by nifedipine or LOE908, and (c) that ET-1-induced MLC phosphorylation and contraction are inhibited by SKF96365 and 2-APB, suggesting that these effects are mainly due to store- and/or receptor Ca 2+ entry.