PHI-1 is one of the newest members of the family of protein phosphatase inhibitor proteins. In isolated enzyme systems, several kinases including PKC and ROCK have been shown to phosphorylate PHI-1. However, it is largely unknown if PHI-1 is phosphorylated in response to agonist stimulation in intact cells. We investigated this question in primary cultured rat aortic vascular smooth muscle cells (VSMCs). With two-dimensional polyacrylamide gel electrophoresis and immunoblot, we found that there are two major PHI-1 spots under resting condition: a minor spot with an acidic isoelectric point (pI) and a major spot with a more alkaline pI. Interestingly, U-46619, a G protein coupled receptor agonist, caused a significant increase in the acidic spot, suggesting that it may represent a phosphorylated form of PHI-1. This was confirmed by phosphatase treatment and by a specific phospho-PHI-1 antibody. Further, we found that angiotensin II, thrombin and U-46619 increased phosphorylated PHI-1 from 9% of total PHI-1 in resting cells to 18%, 18% and 30%, respectively. Furthermore, we found that inhibition of ROCK by Y-27632 or H-1152 selectively diminished U-46619-induced CPI-17 phosphorylation while it did not affect PHI-1 phosphorylation. Activation of ROCK by expressing V14rhoA selectively induced CPI-17 phosphorylation without affecting PHI-1 phosphorylation. On the contrary, inhibition of PKC by GF109203x or by PKC down-regulation selectively diminished U-46619-induced PHI-1 phosphorylation without significantly affecting U-46619-induced CPI-17 phosphorylation. Activating PKC by phorbol 12-myristate 13-acetate (PMA) induced PHI-1 phosphorylation. Taken together, our results first show that agonist induces PHI-1 phosphorylation in VSMCs and divergent kinase signaling couples agonist stimulation to PHI-1 and CPI-17 phosphorylation.