The mechanism or mechanisms by which polymorphonuclear leukocytes (PMN) penetrate junctions between neighboring endothelial cells (EC) to traverse endothelial barriers remain unresolved. We report that chemoattractant-stimulated PMN induce a coordinate increase in both phosphorylation of serine 19 and threonine 18 of EC myosin regulatory light chains and isometric tension generation by EC monolayers. Unstimulated PMN had no effect on either parameter. These findings, coupled with our previous report (Huang et al., J. Cell Biol. 120: 1371-1380, 1993) that chemoattractant-stimulated PMN cause a rise in EC cytosolic free Ca2+, provide strong presumptive evidence that myosin light chain kinase is the EC enzyme responsible for initiating myosin light chain phosphorylation, EC contraction, and isometric tension generation in response to chemoattractant-stimulated PMN. We suggest that, by inducing phosphorylation of EC cytoskeletal proteins, chemoattractant-stimulated PMN induce EC to open their intercellular junctions, thereby facilitating transendothelial movement of these leukocytes.