Effects of ET(B) receptor stimulation and its subcellular pathways were evaluated in carbachol pre-contracted rabbit iris sphincter muscles (n=51). ET(B) stimulation with sarafotoxin (SRTX-c; 10(-10)-10(-6) M) was tested in the absence (n=7) or presence of 10(-5) M of: BQ-788 (ET(B2) receptor antagonist; n=6), L-NA (NOS inhibitor; n=7) or indomethacin (cyclooxygenase inhibitor; n=10). Effects of ET(B) stimulation by endothelin-1 (ET-1; 10(-10)-10(-7) M) in the presence of an ET(A) receptor antagonist (BQ-123; 10(-5) M; n=7) and of ET(B1) stimulation by IRL-1620 (10(-10)-10(-7) M; n=7) were also tested. Finally, the effects of SRTX-c (10(-9)-10(-7) M) in electric field stimulation (EFS) contraction were evaluated (n=7). ET(B) receptor stimulation by SRTX-c or ET-1 in presence of BQ-123 promoted a concentration-dependent relaxation of the rabbit iris sphincter muscle by 10.8+/-2.0% and 9.4+/-1.8%, respectively. This effect was blocked by BQ-788 (-2.3+/-2.0 %), L-NA (4.5+/-2.3 %) or indomethacin (2.3+/-2.9 %). Selective ET(B1) stimulation by IRL-1620 did not relax the iris sphincter muscle (0.9+/-5.4 %). EFS elicited contraction was not altered by SRTX-c. In conclusion, ET(B) receptor stimulation relaxes the carbachol precontracted iris sphincter muscle, an effect that is mediated by the ET(B2) receptor subtype, through NO and the release of prostaglandins.