Abstract The activities of glutathione peroxide, superoxide dismutase and catalase, enzymes which play a critical role in protection of the vascular endothelium from oxygen free-radical injury, were determined in large vessel endothelial cells obtained under three different growth conditions: (1) from freshly isolated from bovine pulmonary arteries, (2) in the first (primary) subculture and (3) after six serial subcultures (6.5 population doublings). The endothelium was obtained by mechanically scraping the vascular lumen. Endothelial cell monolayers were detached mechanically from the substratum prior to passage. No proteolytic enzymes were used in either procedure. The activities of catalase, superoxide dismutase and glutathione peroxidase determined in freshly isolated endothelial cells were, respectively, 39.9 ± 10.3, 2.2 ± 0.8 and 3.0 ± 0.5 × 10 −2 units per mg protein. After primacy culture there was no change in superoxide dismutase activity, but a significant decrease in glutathione peroxidase activity to 1.4 ± 0.4 × 10 −2 was observed, and catalase activity dropped significantly to 18.6 ± 5.0 units per mg protein. After 6.5 population doublings, the activity of all three enzymes returned to values similar to those of the freshly isolated cells. A fourfold increase in the protein to DNA ratio occurred in cells in primary culture and was maintained in sixth-passage cells. This increase in endothelial cell size upon culture was reflected in the electron microscopic evidence of cellular hypertrophy. Measurement of the rate of transport of 5-hydroxytryptamine by endothelial cell monolayers revealed a substantial loss upon multiple passage. Transport in the sixth-passage cells was decreased to one-half the rate of primary cells. Comparison of the ultrastructural characteristics of cells from the three groups showed more endomembrane structure and higher numbers of mitochondria in the passaged cells, suggesting that passaged cells were more metabolically and synthetically active than in situ cells. In contrast, in situ cells showed more elaborate cell junction complexes than the passaged cells. The observed differences in antioxidant enzyme content, function and morphology between endothelial cells in situ and in culture underscore the need for careful characterization of cultured cells before their use as in vitro models for the vascular endothelium.