Heparin inhibits vascular smooth muscle cell (VSMC) proliferation, but mechanisms remain elusive. Because heparin inhibits signaling through multiple kinase cascades, we investigated the possibility that phosphatases could be involved. Mitogen-activated protein kinase phosphatase-1 (MKP-1) was the predominant MKP detected in VSMC lines. MKP-1 protein was increased by serum stimulation of quiescent cells, and this increase was diminished by heparin (1 microg/mL). Increased MKP-1 expression was dependent on the mitogen-activated protein kinase, Erk. Decreased Erk activity in the presence of heparin preceded, and may account for, decreased MKP-1. The antimitogenic effects of heparin are therefore unlikely to act through a shift in the kinase/phosphatase balance, but rather through direct kinase suppression. However, because MKP-1 is known to cause an increase in activity of kinases upstream of Erk, that may signal through additional pathways, the decrease in MKP-1 activity may paradoxically enhance heparin's antiproliferative effects. VSMC selected to grow in the presence of heparin express decreased levels of MKP-1 that are unresponsive to heparin, and Erk activity becomes unresponsive to heparin in one cell line. We conclude that phosphatase activation is not a direct mechanism of suppression of multiple kinase cascades by heparin.