The objective of this study was to determine the effect of pulsatile flow on endothelin (ET) receptor expression in vascular smooth muscle cells (VSMC) cocultured with endothelial cells (EC). Using a perfused transcapillary coculture system which permits the chronic exposure of cultured EC and VSMC to physiological shear stresses, cocultures were exposed to stepwise increases in flow up to (1) 2 ml/min (low flow: 0.5 dyn/cm<sup>2</sup>) or (2) 44 ml/min (high flow: 15 dyn/cm<sup>2</sup>) and maintained for 72 h before SMC and EC were harvested separately. There was a significant increase in [<sup>125</sup>I]-ET-1 binding in cocultured VSMC exposed to high flow as compared to low flow (B<sub>max</sub>: 75 ± 22 vs. 152 ± 10 fmol [<sup>125</sup>I]ET-1 bound/mg protein) in the absence of any change in the affinity (K<sub>D</sub>) of ET-1 for its receptor. ET-1 peptide mRNA levels were significantly decreased in EC exposed to high flow. The increase in [<sup>125</sup>I]ET-1 binding was associated with an increase in ET-A and ET-B receptor mRNA levels and was EC dependent as [<sup>125</sup>I]ET-1 binding in monocultured VSMC was the same, regardless of flow conditions. However, the amount of [<sup>125</sup>I]ET-1 binding on VSMC cultured in the absence of EC was significantly greater than that on cocultured VSMC. High flow caused a significant increase in endothelial nitric oxide synthase (NOS) activity in EC and prostacyclin levels in the perfusing medium. Flow-mediated upregulation of ET receptors was diminished by treatment with N<sup>G</sup>-nitro-L-arginine-methyl ester, a NOS inhibitor, whereas indomethacin, a cyclooxygenase inhibitor, had no significant effect. Collectively, these data suggest that flow-induced changes in ET receptor expression in VSMC are endothelium dependent and are in part mediated by nitric oxide. Modulation of ET receptor expression by EC may thus represent an important mechanism whereby hemodynamic forces regulate vessel wall function.