Hemodynamic regulation of the endothelial gap junction protein connexin43 (Cx43) was studied in a model of controlled disturbed flows in vitro. Cx43 mRNA, protein expression, and intercellular communication were mapped to spatial variations in fluid forces. Hemodynamic features of atherosclerotic lesion-prone regions of the vasculature (flow separation and recirculation) were created for periods of 5, 16, and 30 h, with laminar shear stresses ranging between 0 and 13.5 dynes/cm². Within 5 h, endothelial Cx43 mRNA expression was increased in all cells when compared with no-flow controls, with highest levels (up to 6- to 8-fold) expressed in regions of flow recirculation corresponding to high shear stress gradients. At 16 h, Cx43 mRNA expression remained elevated in regions of flow disturbance, whereas in areas of fully developed, undisturbed laminar flow, Cx43 expression returned to control levels. In all flow regions, typical punctate Cx43 immunofluorescence at cell borders was disrupted by 5 h. After 30 h of flow, disruption of gap junctions persisted in cells subjected to flow separation and recirculation, whereas regions of undisturbed flow were substantially restored to normal. These expression differences were reflected in sustained inhibition of intercellular communication (dye transfer) throughout the zone of disturbed flow (84.2 and 68.4% inhibition at 5 and 30 h, respectively); in contrast, communication was fully reestablished by 30 h in cells exposed to undisturbed flow. Up-regulation of Cx43 transcripts, sustained disorganization of Cx43 protein, and impaired communication suggest that shear stress gradients in regions of disturbed flow regulate intercellular communication through the expression and function of Cx43.