Myosin phosphatase (MLCP) plays a critical regulatory role in the Ca(2+) sensitivity of myosin phosphorylation and smooth muscle contraction. It has been suggested that phosphorylation at Thr696 of the MLCP regulatory subunit (MBS/MYPT1) and at Thr38 of the CPI-17, which is MLCP inhibitor protein, results in inhibition of MLCP activity. The phosphorylation of CPI-17 Thr38 is thought to play an important role in G-protein-mediated inhibition of MLCP in tonic arterial smooth muscle. The CPI-17 phosphorylation transiently increased after agonist stimulation in both alpha-toxin skinned and intact fibers. The time course of the increase in CPI-17 phosphorylation after stimulation correlated with the increase in myosin regulatory light chain (MLC20) phosphorylation. These results strongly suggest that the phosphorylation of CPI-17 plays a significant role in the agonist-induced increase in myosin phosphorylation and contraction of smooth muscle in the Ca(2+)-independent activation mechanism of smooth muscle contraction. This anti-Phospho-CPI-17 Thr38 monoclonal antibody has been validated with PKC, however it has the potential for use in evaluating other serine threonine kinases such as Rho-Kinase, protein kinase N and ILK.