Smooth muscle relaxation in response to NO signaling is due, in part, to a Ca(2+)-independent activation of myosin light chain (MLC) phosphatase by protein kinase G Iα (PKGIα). MLC phosphatase is a trimeric complex of a 20-kDa subunit, a 38-kDa catalytic subunit, and a 110-133-kDa myosin-targeting subunit (MYPT1). Alternative mRNA splicing produces four MYPT1 isoforms, differing by the presence or absence of a central insert and leucine zipper (LZ). The LZ domain of MYPT1 has been shown to be important for PKGIα-mediated activation of MLC phosphatase activity, and changes in LZ+ MYPT1 isoform expression result in changes in the sensitivity of smooth muscle to NO-mediated relaxation. Furthermore, PKGIα has been demonstrated to phosphorylate Ser-694 of MYPT1, but phosphorylation at this site does not always accompany cGMP-mediated smooth muscle relaxation. This study was designed to determine whether MYPT1 isoforms are differentially phosphorylated by PKGIα. The results demonstrate that purified LZ+ MYPT1 fragments are rapidly phosphorylated by PKGIα at Ser-667 and Ser-694, whereas fragments lacking the LZ domain are poor PKGIα substrates. Mutation of Ser-667 and Ser-694 to Ala and/or Asp showed that Ser-667 phosphorylation is more rapid than Ser-694 phosphorylation, suggesting that Ser-667 may play an important role in the activation of MLC phosphatase. These results demonstrate that MYPT1 isoform expression is important for determining the heterogeneous response of vascular beds to NO and NO-based vasodilators, thereby playing a central role in the regulation of vascular tone in health and disease.