MAP kinase (ERK1 and ERK2) regulates cell motility by phosphorylating myosin light chain kinase (MLCK), which enhances myosin light chain (MLC) phosphorylation, leading to cell migration on the extracellular matrix. Cell adhesion and migration on the extracellular matrix are critical for processes such as angiogenesis, inflammation, embryonic development, wound healing, and tumor metastasis. Integrin-mediated adhesion to the extracellular matrix activates focal adhesion kinase and downstream MAP kinase pathways, which control gene transcription and cell proliferation. However, MAP kinase can directly activate intracellular motility machinery without de novo gene transcription. Mutationally active MAP kinase kinase (MEK) activates MAP kinase, leading to MLCK and MLC phosphorylation, and enhanced cell migration. In vitro experiments show that ERK-phosphorylated MLCK has increased capacity to phosphorylate MLC and is more sensitive to calmodulin. These findings define a signaling pathway downstream of MAP kinase that influences cell migration on the extracellular matrix. MAP kinase activation is critical for cell migration but not adhesion or spreading on collagen. Inhibition of MEK activity reduces cell migration but does not affect adhesion or spreading. Mutationally active MEK1 enhances cell migration on collagen, fibronectin, and vitronectin. MLCK, a key regulator of MLC phosphorylation, is phosphorylated by MAP kinase, leading to increased MLCK activity and sensitivity to calcium/calmodulin. MLCK activity is required for MAP kinase-induced cell migration, as shown by experiments using a dominant-negative MLCK mutant. These results suggest that MAP kinase activates the cell's motility machinery by enhancing MLCK activity, leading to increased MLC function and cell movement. The study provides evidence that MAP kinase signaling can regulate cell migration independently of gene transcription, highlighting its role in cell motility.MAP kinase (ERK1 and ERK2) regulates cell motility by phosphorylating myosin light chain kinase (MLCK), which enhances myosin light chain (MLC) phosphorylation, leading to cell migration on the extracellular matrix. Cell adhesion and migration on the extracellular matrix are critical for processes such as angiogenesis, inflammation, embryonic development, wound healing, and tumor metastasis. Integrin-mediated adhesion to the extracellular matrix activates focal adhesion kinase and downstream MAP kinase pathways, which control gene transcription and cell proliferation. However, MAP kinase can directly activate intracellular motility machinery without de novo gene transcription. Mutationally active MAP kinase kinase (MEK) activates MAP kinase, leading to MLCK and MLC phosphorylation, and enhanced cell migration. In vitro experiments show that ERK-phosphorylated MLCK has increased capacity to phosphorylate MLC and is more sensitive to calmodulin. These findings define a signaling pathway downstream of MAP kinase that influences cell migration on the extracellular matrix. MAP kinase activation is critical for cell migration but not adhesion or spreading on collagen. Inhibition of MEK activity reduces cell migration but does not affect adhesion or spreading. Mutationally active MEK1 enhances cell migration on collagen, fibronectin, and vitronectin. MLCK, a key regulator of MLC phosphorylation, is phosphorylated by MAP kinase, leading to increased MLCK activity and sensitivity to calcium/calmodulin. MLCK activity is required for MAP kinase-induced cell migration, as shown by experiments using a dominant-negative MLCK mutant. These results suggest that MAP kinase activates the cell's motility machinery by enhancing MLCK activity, leading to increased MLC function and cell movement. The study provides evidence that MAP kinase signaling can regulate cell migration independently of gene transcription, highlighting its role in cell motility.