Myosin phosphatase (MLCP) plays a critical regulatory role in the Ca2+ sensitivity of myosin phosphorylation and smooth muscle contraction. It has been suggested that phosphorylation at Thr695 of the MLCP regulatory subunit (MYPT1) and at Thr38 of the MLCP inhibitor protein CPI-17 results in inhibition of MLCP activity. We have previously demonstrated that CPI-17 Thr38 phosphorylation plays an important role in G-protein-mediated inhibition of MLCP in tonic arterial smooth muscle. Here, we attempted to evaluate the function of MYPT1 in phasic rabbit portal vein (PV) and vas deferens (VD) smooth muscles. Using site- and phospho-specific antibodies, phosphorylation of MYPT1 Thr695 and CPI-17 Thr38 was examined along with MYPT1 Thr850, which is a non-inhibitory Rho-kinase site. We found that both CPI-17 Thr38 and MYPT1 Thr850 were phosphorylated in response to agonists or GTPγS concurrently with contraction and myosin phosphorylation in α-toxin-permeabilized PV tissues. In contrast, phosphorylation of MYPT1 Thr695 did not increase. Comparable results were also obtained in both permeabilized and intact VD. The Rho-kinase inhibitor Y-27632 and the protein kinase C (PKC) inhibitor GF109203X suppressed phosphorylation of MYPT1 Thr850 and CPI-17 Thr38, respectively, in intact VD while MYPT1 Thr695 phosphorylation was insensitive to both inhibitors. These results indicate that phosphorylation of MYPT1 Thr695 is independent of stimulation of G-proteins, Rho-kinase or PKC. In the phasic PV, phosphorylation of CPI-17 Thr38 may contribute towards inhibition of MLCP while the phasic visceral VD, which has a low CPI-17 concentration, probably utilizes other Ca2+ sensitizing mechanisms for inhibiting MLCP besides phosphorylation of MYPT1 and CPI-17.