The small molecular mass heat shock protein of 27 kDa (HSP27) has been shown to influence actin filament dynamics and endothelial cell behavior in ways similar to those observed during laminar flow. We have employed human umbilical vein endothelial cells to determine whether fluid shear stress affects HSP27 expression or phosphorylation. After a shear stress of 16 dyn/cm2, HSP27 became more highly phosphorylated, with maximum increase in phosphorylation levels (3-fold) attained by 30 min and sustained for at least 20 h. HSP27 antigen levels did not change; however, HSP27 mRNA levels decreased by 20% after 16 h. In bovine aortic endothelial cells stably transfected with the wild-type human HSP27 gene, shear stress induced the phosphorylation of both the exogenous human HSP27 and the endogenous bovine HSP25. The product of a transfected mutant HSP27 gene in which the putative phosphorylation sites Ser-15, Ser-78, and Ser-82 had been replaced with Gly was not phosphorylated. Thus the modulation of HSP27 and its activity by shear stress is mediated through a posttranslational mechanism and differs from the shear stress induction of immediate early genes at the level of transcription.