Endothelial cells from different tissues display variations in morphology, intercellular junctions, cell surface and growth properties, and in production of basal lamina components, both in vivo and in vitro. We have investigated the spectra of extracellular proteins secreted by bovine endothelial cells cultured from large vessels, cornea, and capillaries. Aortic, venous, and corneal endothelial cells displayed highly similar patterns of protein synthesis as judged by analysis of the culture medium; the major products were fibronectin, a glycoprotein similar or identical to platelet thrombospondin, and Type III procollagen. Ion-exchange chromatography, followed by peptide mapping, confirmed the presence of EC, a novel endothelial collagen previously described in bovine aortic endothelial cell cultures. Minor variations were found in the collagens of the cell layers: Type III, the predominant interstitial collagen, was associated with the basement membrane Types IV and V and, in the case of corneal endothelium, with Type I. In contrast, capillary endothelial cells secreted significantly more collagen than did the aortic, venous, and corneal cells. Approximately 50% of the protein in the culture medium was collagenous and consisted of Types I and III collagen in a ratio of 2:3. These interstitial collagens were the only types detected in capillary cell layers as well. The pattern and overall rate of collagen synthesis by capillary endothelial cells in vitro contrasted significantly with that of the other endothelial cell types and closely resembled that described for cultures of sprouting endothelium. These alterations in secretory phenotype may reflect: 1) a true difference in cell type between capillary and other types of endothelium, 2) differences resulting from cell isolation and initial culture conditions, or 3) a correlation between growth regulation and protein synthesis.