The present study was designed to examine the effect of thrombin on endothelin production by cultured human umbilical vein endothelial cells (HUVEC) by radioimmunoassay. Cultured HUVEC released immunoreactive endothelin-1 (ir-endothelin-1) into the medium in a time-dependent manner. This release was inhibited completely by 10 micrograms/mL cycloheximide and is, therefore, directly related to de novo protein synthesis. Further endothelin release was stimulated by the addition of thrombin to the culture medium. Thrombin-induced ir-endothelin-1 release also was inhibited completely by 10 micrograms/mL cycloheximide. These results suggest that HUVEC in culture produce ir-endothelin-1 slowly, but continuously, and that this production is stimulated by thrombin.