Tonic physiological activity of RhoA/Rho-kinase contributes to the maintenance of penile flaccidity through its involvement in the Ca²⁺ sensitization of erectile tissue smooth muscle. The present study hypothesized that Rho-kinase is also involved in the modulation of Ca²⁺ entry in rat penile arteries. Arterial segments were mounted in microvascular myographs for simultaneous measurements of intracellular Ca²⁺ [Ca²⁺]_i and force. The Rho-kinase inhibitor Y-27632 markedly reduced noradrenaline-mediated electrically-induced contractions and the increases in both [Ca²⁺]_i and tension elicited by the ₁-adrenoceptor agonist phenylephrine (Phe). In contrast, the protein kinase C (PKC) inhibitor Ro 31-8220 reduced tension without altering the Phe-induced [Ca²⁺]_i. In the presence of nifedipine, Y-27632 still inhibited the non L-type Ca²⁺ signal and blunted Phe contraction. Y-27632 did not impair the capacitative Ca²⁺ entry evoked by store depletion with cyclopiazonic acid (CPA), but largely reduced the Ba²⁺ influx stimulated by Phe in Fura-2-AM loaded arteries. Western Blot analysis of rat penile arteries showed the expression of TRPC6 proteins. Addition of Y-27632 to arteries depolarized with high KCl markedly reduced tension without changing [Ca²⁺]_i. In -toxin-permeabilized penile arteries stimulated with threshold Ca²⁺ concentrations, Y-27632 inhibited the sensitization induced by either GTP--S or Phe in the presence of GTP--S. However, Y-27632 failed to alter contractions induced by a maximal concentration of free Ca²⁺. These results suggest that Rho-kinase, besides its contribution to the Ca²⁺ sensitization of the contractile proteins, is also involved in the regulation of Ca²⁺ entry through a non-selective cation channel activated by ₁-adenoceptor stimulation in rat penile arteries.