Endothelial cell (EC) contraction results in intercellular gap formation and loss of the selective vascular barrier to circulating macromolecules. We tested the hypothesis that phosphorylation of regulatory myosin light chains (MLC) by Ca²⁺/calmodulin‐dependent myosin light chain kinase (MLCK) is critical to EC barrier dysfunction elicited by thrombin. Thrombin stimulated a rapid (<15 sec) increase in [Ca²⁺]_i which preceded maximal MLC phosphorylation (60 sec) with a 6 to 8‐fold increase above constitutive levels of phosphorylated MLC. Dramatic cellular shape changes indicative of contraction and gap formation were observed at 5 min with maximal increases in albumin permeability occurring by 10 min. Neither the Ca²⁺ ionophore, A23187, nor phorbol myristate acetate (PMA), a direct activator of protein kinase C (PKC), alone or in combination, produced MLC phosphorylation. The combination was synergistic, however, in stimulating EC contraction/gap formation and barrier dysfunction (3 to 4‐fold increase). Down‐regulation or inhibition of PKC activity attenuated thrombin‐induced MLC phosphorylation (∼40% inhibition) and both thrombin‐ and PMA‐induced albumin clearance (∼50% inhibition). Agents which augmented [cAMP]_i partially blocked thrombin‐induced MLC phosphorylation (∼50%) and completely inhibited both thrombin‐ and PMA‐induced EC permeability (100% inhibition). Furthermore, cAMP produced significant reduction in the basal levels of constitutive MLC phosphorylation. Finally, MLCK inhibition (with either ML‐7 or KT 5926) or Ca²⁺/calmodulin antagonism (with either trifluoperazine or W‐7) attenuated thrombin‐induced MLC phosphorylation and barrier dysfunction. These results suggest a model wherein EC contractile events, gap formation and barrier dysfunction occur via MLCK‐dependent and independent mechanisms and are significantly modulated by both PKC and cAMP‐dependent protein kinase A activities. © 1995 Wiley‐Liss, Inc.