In endothelial cells (EC), caveolae or plasmalemmal vesicles (PVs) represent a structurally and biochemically specialized membrane microdomain. Since few data are available on the biochemical composition of PVs of large vessel endothelium, we have designed experiments to isolate this domain and to analyze its chemical components. A highly purified apical membrane fraction was obtained from cultured bovine aortic EC by using cationic colloidal silica (silica-ap), or the EC were surface-radioiodinated and a cell homogenate was prepared. Detergent treatment (Triton X-100; TX) and mechanical disruption of both the silica-ap fraction and cell homogenate followed by ultracentrifugation on a sucrose gradient gave detergent-soluble and detergent-insoluble membranous fractions. The lowest density TX-insoluble fraction appeared morphologically as distinct vesicles (caveolae; 60 nm average diameter; PVs fraction). Biochemical characterization of the PVs fraction (by comparison with the soluble fraction) revealed the presence, at high concentration, of specific caveolar markers, viz., caveolin (both isoforms, the 24-kDa form being conspicuously more abundant) and Ca2+-ATPase. By contrast, angiotensin-converting enzyme and alkaline phosphodiesterase were present almost exclusively in the TX-soluble fraction. The glycoproteins in the PVs fraction were of apparent molecular weights 52, 68, 95, and 114 kDa. Analysis of the fatty acid composition revealed more palmitoleic and stearic acid in the PVs fraction then in the TX-soluble fraction. Thus, in comparison with the plasmalemma proper, the PVs fraction (1) is detergent-insoluble; (2) contains caveolin in two isoforms; (3) contains Ca2+-ATPase at high concentration; (4) contains a set of specific glycoproteins; and (5) is enriched in palmitoleic and stearic acids.