Bovine aortic endothelium has been examined with respect to the synthesis of coagulation factor V. After cultured cells reached confluency, samples of supernatant culture media and solubilized cells were analyzed for factor V in a two-stage bioassay and in a double- antibody radioimmunoassay. In addition, preconfluent cells were pulsed for 4 days with 35S-methionine in methionine-free media. After the 4- day pulse, supernatant media were chromatographed on a factor V monoclonal antibody-Sepharose resin to isolate 35S-labeled factor V. The isolated material and 125I-factor V standards were analyzed by electrophoresis and autoradiography. The bioassay indicated an increase, with time, of unactivated factor V in the culture supernatant, whereas solubilized cells were negative for factor V. The radioimmunoassay indicated an increase, with time, of factor V antigen in the culture supernatants, and the solubilized cells yielded a constant level of antigen per cell. Autoradiograms of electrophoretograms of immunoadsorbed 35S-culture supernatant with 125I- factor V/Va standards revealed labeled proteins with electrophoretic mobilities compatible with 125I-factor V/Va standards. The data obtained from three different sources-bioassay, radioimmunoassay, and 35S-methionine incorporation-all indicate that factor V is synthesized by cultured bovine aortic endothelium.