Delayed cerebral vasospasm after subarachnoid hemorrhage (SAH) may be due, in part, to altered regulation of arterial smooth muscle contraction. Contraction of cerebral arteries to serotonin is augmented after experimental SAH. We hypothesized that activation of rho-associated kinase (rho-kinase) contributes to augmented contraction of cerebral arteries to serotonin after SAH. Autologous arterial blood (SAH) or artificial cerebrospinal fluid (control) was injected into the cisterna magna of anesthetized rabbits. Two days after injection, the basilar artery was excised and isometric contraction of arterial rings was recorded. In SAH compared to control rabbits, maximum contraction of the basilar artery to serotonin was augmented about 4-fold (P<0.01). Contraction to histamine was similar in the two groups. Fasudil hydrochloride (3 µmol/l), an inhibitor of rho-kinase, markedly attenuated serotonin-induced contraction. Fasudil had little effect on contractions induced by histamine or phorbol 12,13-dibutylate. In addition, phosphorylation of myosin phosphatase, a major target of rho-kinase in regulation of smooth muscle contraction, in the basilar artery was examined by Western blotting. In basilar arteries of SAH but not control rabbits, serotonin increased phosphorylation of myosin phosphatase about 2-fold at Thr853 of the myosin-targeting subunit. These results suggest that enhanced activation of rho-kinase contributes to augmented contraction of the basilar artery to serotonin after SAH.