Myosin light chain phosphorylation results in cellular contraction and is a critical component of agonist-mediated endothelial cell (EC) junctional gap formation and permeability. We have shown that this reaction is catalyzed by a novel high molecular-weight Ca²⁺/calmodulin-dependent nonmuscle myosin light chain kinase (MLCK) isoform recently cloned in human endothelium (Am. J. Respir. Cell Mol. Biol., 1997;16:489–494). To characterize EC MLCK expression further in cultured and adult tissues, we employed immunoblotting techniques and reverse transcriptase–polymerase chain reaction to demonstrate that freshly isolated and cultured human macro- and microvascular EC express only the EC MLCK isoform (214 kD), which is distinct from smooth-muscle MLCK isoforms (130 to 150 kD). Immunocytochemical studies demonstrated the presence of the high molecular-weight MLCK isoform in adult human cardiac endothelium using anti-MLCK antibodies, which preferentially recognize the high molecular-weight EC MLCK isoform. Monitoring of MLCK expression in different cell types with antibodies generated against a unique human EC MLCK N-terminal sequence revealed a high level of expression of the 214-kD enzyme in endothelium, minimal level of expression in smooth muscle, and no expression in skeletal muscle. These data suggest that the novel 214-kD kinase, the only MLCK isoform found in endothelium, may be preferentially expressed in this nonmuscle tissue.