Cytosolic Ca²⁺ ([Ca²⁺];) plays an important role in endothelial cell signaling. Although it has been suggested that the influx of Ca²⁺ can be triggered by depletion of intracellular Ca²⁺ stores, the mechanism (or mechanisms) underlying this phenomenon needs further elaboration. In the present study, involvement of myosin light‐chain kinase (MLCK) in the regulation of Ca²⁺ signaling was investigated in agonist‐ and fluid flow‐stimulated endothelial cells loaded with Ca²⁺‐sensitive dyes. Bradykinin (BK) and thapsigargin caused an increase in [Ca²⁺]_i followed by a sustained rise due to Ca²⁺ influx from extracellular space and shifted total myosin light‐chain (MLC) from the unphosphorylated to the diphosphorylated form. ML‐9 (100 pM), an inhibitor of MLCK, abolished Ca²⁺ influx and prevented MLC diphosphorylation in BK‐ and thapsigargin‐treated cells, but did not affect Ca²⁺ mobilization from internal stores. Fluid flow stimulation (shear stress=5 dynes/cm²) increased [Ca²⁺]_i and enhanced MLC phosphorylation. ML‐9 also inhibited Ca²⁺ response and MLC phosphorylation in fluid flow‐stimulated cells. The Ca²⁺ influx in response to BK was linearly correlated with the diphosphorylation of MLC in ML‐9 treated cells. Effects of ML‐5 and ML‐7, analogs of ML‐9, to inhibit Ca²⁺ influx paralleled their potencies to inhibit MLCK activity. These findings demonstrate that MLCK plays an essential role in regulating the plasmalemmal Ca²⁺ influx in agonist‐ and fluid flow‐stimulated endothelial cells. This study is the first to report the close relationship between Ca²⁺ influx and MLC diphosphorylation.—Watanabe, H., Takahashi, R., Zhang, X.‐X., Goto, Y., Hayashi, H., Ando, J., Isshiki, M., Seto, M., Hidaka, H., Niki, I., Ohno, R. An essential role of myosin light‐chain kinase in the regulation of agonist‐ and fluid flow‐stimulated Ca²⁺ influx in endothelial cells. FASEB J. 12, 341–348 (1998)