Noradrenaline (NA) elicits robust and sustained contractions of rat vas deferens in Ca²⁺-free/EGTA medium (Ashoori & Tomita, 1983). Agents that deplete intracellular Ca²⁺ stores or block its release or protein kinase C activation do not inhibit the Ca²⁺-free contraction (Amobi et al., 1999). In order to elucidate the excitation-contraction coupling pathway underlying the Ca²⁺-free response and the possible role of Rho kinase-mediated Ca²⁺sensitization, the effects of Rho-kinase inhibitors were investigated. The contribution of Ca²⁺sensitization to Ca²⁺-dependent contractions of both rat and human epididymal vas deferens were also evaluated. Male Sprague Dawley rats were killed humanely. Epididymal portions of isolated rat vas deferens, longitudinal muscle strips and rings of circular muscle from human vasectomy specimens (patient consent and College ethical approval obtained) were set up for tension recordings and superfused (2.5 ml min-1) with Krebs’-bicarbonate solution (36 oC). NA-induced Ca²⁺-free contraction of rat vas deferens was relaxed dose-dependently by Rho kinase inhibitors, Y-27632 (0.1-10 μM in different experiments, Fig. 1A) or HA 1077 (not shown). IC50 values (mean ± S.E.M.) were, respectively, 1.08 ± 0.13 μM (n=10) and 1.75 ± 0.43 μM (n=8) respectively. Exposure to Y-27632 (0.01-10 μM in different experiments) did not change basal tone but Y-27632 (0.01-3 μM) reduced the maximum of concentration-response curves to NA (0.1-300 μM) in Ca²⁺-free medium and Y-27632 (10 μM) abolished the response. In contrast, the NA response curve was unaffected by MLCK inhibitors, ML-9 (1-3μM) or wortmannin (1-3 μM) but the maximum was depressed by ML-9 (10 μM) or wortmannin (10 μM). Contractions of rat and human (longitudinal or circular muscle) vas deferens evoked in normal Krebs’ (Ca²⁺, 2.5 mM) by NA (100 μM) or high [K+]_o (120 mM) were reliably depressed but not abolished by Y-27632 (1-10 μM). Abolition required the combination of Y-27632 (10 μM) and ML-9 (30 μM). These results suggest that Rho kinase-mediated Ca²⁺sensitization is the major mechanism underlying NA-induced Ca²⁺-free contraction of rat vas deferens and contributes to Ca²⁺-dependent contractions of both rat and human vas deferens.