Integrin-mediated substrate adhesion of endothelial cells leads to intracellular signaling, including the activation of ERK 1/2 (extracellular regulated kinases 1 and 2), members of the mitogen-activated protein kinase (MAPK) family. MKP-1 is a dual-specificity protein phosphatase that may play an important role in regulating MAPK activity through dephosphorylation of threonine and tyrosine. Adhesion of human umbilical vein endothelial cells to fibronectin increased MKP-1 protein and mRNA levels, which reached a maximum at 60 min, while MAPK activity was maximal at 30 min. The MEK inhibitor PD98059 blocked activation of MAPK as well as the induction of MKP-1 during adhesion. The transcription inhibitor actinomycin D blocked MKP-1 induction and produced prolonged MAPK activation during adhesion. In contrast, endothelial adhesion to poly-L-lysine did not alter MAPK activity or MKP-1 levels. These findings demonstrate that integrin-mediated adhesion of endothelial cells to fibronectin results in transcriptional activation of MKP-1 through a MAPK-dependent mechanism. Regulation of MKP-1 by MAPK likely represents an important negative-feedback mechanism.