We established culture lines of porcine endothelial cells derived from both the aortic valve and the ascending aorta. These cells had the typical morphology of vascular endothelial cells in culture and contained angiotensin-converting enzyme activity. The cells derived from the aorta grew well in standard tissue culture dishes; the valvular endothelial cells, however, could not be passed serially in vitro unless cultivated in dishes precoated with fibronectin. Moreover, under the conditions used, the valvular cells showed substantial changes in their morphology when deprived of fibronectin coating for several passages but reestablished typical vascular endothelial morphology when subcultured to fibronectin-coated dishes. The aortic cells did not show this behavior. Endogenous labeling of proteins synthesized in vitro by the two cell types with 35S-methionine revealed that a high molecular weight protein produced in substantial quantities by the aortic cells was synthesized only in trace amounts by the valvular cells. This protein could be isolated by gelatin-agarose chromatography and showed an apparent molecular weight under reduced conditions of 235,000 to 245,000 on sodium dodecyl sulfate gel electrophoresis. Synthesis of gelatin-binding proteins, an indirect estimation of fibronectin production, as well as direct measurement of porcine fibronectin present in the culture supernatants using an immunoradiometric assay, showed that aortic endothelial cells synthesized and released 10-fold more fibronectin in vitro than did valvular endothelial cells. We suggest that this relative deficiency of in vitro fibronectin synthesis by valvular cells, if paralleled in vivo, may have important implications for the pathogenesis of lesions unique to the cardiac valve.