Biochemical studies have shown that wortmannin is an inhibitor of myosin light chain (MLC) kinase (Nakanishi et al. (1992) J. Biol. Chem. 267: 2157-2163). To investigate the role of MLC kinase in smooth muscle contractions, we examined the effects of wortmannin on isolated smooth muscles of the rat aorta. Wortmannin (1 ~tM) decreased MLC phosphorylation and the amplitude of contractions induced by high K Γ· (72.7 raM) to a level seen at rest. This occurred without a change in cytosolic Ca 2Γ· levels ([Ca2+]0. In contrast, wortmannin only partially inhibited the sustained contractions induced by phenylephrine (1 gM) and prostaglandin F2~ (PGF2~, 10 gM) without a change in the [Ca2+]~. On the other hand, wortmannin (1 or 10 gM) reduced the increase in MLC phosphorylation induced by phenylephrine and PGF2~ to a level seen at rest. In the absence of external Ca 2 +, caffeine (20 raM) induced a transient increase in [Ca2+]i and force with an increase in MLC phosphorylation. Wortmannin completely inhibited the increase in MLC phosphorylation and contraction induced by caffeine without affecting the increase in [-Ca2+li . In the absence of external Ca 2+, phenylephrine induced a small transient increase in [-Ca2+]i, MLC phosphorylation and generation of force. This was followed by a small sustained contraction without an increase in [Ca2+li and MLC phosphorylation. Wortmannin (1 gM) inhibited the transient phase of the contraction and the increase in MLC phosphorylation without affecting the transient increase in [Ca 2 +li nor the sustained contraction. Wortmannin inhibited the Ca2Γ·-induced contraction in permeabilized rat mesenteric artery, although it did not inhibit the