Peroxisome proliferator-activated receptor (PPAR) ligands, thiazolidinediones (TZD), have been demonstrated to regulate vascular reactivity. We examined the effect of pioglitazone (PIO - 20µM) in rat primary cultured aortic smooth muscle cells on constitutive phosphorylation of the regulatory subunit (MYPT) of myosin phosphatase. PIO decreased phosphoyrlation of Thr697 on MYPT within 15 min. and the inhibition was maintained up to 6 hours. The PPAR antagonist GW9662 (5µM) abrogated the inhibition of Thr697 phosphorylation mediated by PIO. Because longer term PIO treatment inhibits RhoA/Rho-kinase signaling and Thr697 phosphorylation we tested the effect of the Rho-kinase inhibitor Y27632 (10µM) on the inhibition of Thr697 phosphorylation by PIO. Y27632 alone inhibited Thr697 phosphorylation and there was an additive effect with PIO. In addition, up to 1h of PIO treatment did not affect RhoA localization, or decrease ROCK-dependent phosphorylation of Thr855. These results suggest that the effect of PIO is independent of inhibition of RhoA/Rho-kinase. PIO increased the phosphorylation of Ser696 in the same time course as its effect on Thr697. Ser696 has been shown to be phosphorylated by PKA and PKG. The protein kinase A (PKA) H89 (10µM) and protein kinase G (PKG) KT5823 (0.5µM) inhibitors abrogated the effect of PIO on both Thr697 and Ser696 phosphorylation. The constitutive turnover of phosphorylation of Thr697 is rapid, suggesting that the decreased phosphorylation of Thr697 by PIO is due to enhanced phosphorylation of Ser696. This is supported by the finding that PIO blocks AngII stimulated phosphorylation of Thr697, but not AngII stimulated RhoA translocation. Therefore, the effect of shorter term PIO apparently is to increase MLCP activity thereby desensitizing the vascular smooth muscle to agonist signaling.