Vascular endothelial cells (EC) are the initial cells within the vascular wall exposed to decreases in blood ambient oxygen concentration. The mechanisms by which they tolerate low levels of oxygen are unknown, but may parallel the response to other cellular stresses, such as heat shock. After 4-8 h of hypoxia, we found a decrease in total protein synthesis in both cultured bovine aortic and pulmonary arterial EC. SDS-PAGE and autoradiographic analysis of [35S]methionine-labeled proteins demonstrated the concomitant induction of a specific set of proteins (Mr 34, 36, 47, and 56 kD) in both cell types. These hypoxia-associated proteins (HAPs) were cell-associated and up-regulated in a time- and oxygen concentration-dependent manner. Comparison of these proteins with heat shock proteins (HSPs) demonstrated that HAPs were distinct from HSPs. EC maintained chronically in 3% O2 continued to synthesize elevated levels of HAPs, yet further up-regulated these proteins when exposed to 0% O2. The presence of five times the normal media glucose concentration did not alter the appearance of HAPs. Hypoxia sensitive renal tubular epithelial cells up-regulated no proteins corresponding to HAPs and were irreversibly damaged within 8 h of exposure to 0% O2. In vitro translation experiments demonstrated that the steady-state level of several mRNAs was higher in the anoxic EC than in normoxic EC and encoded for proteins of Mr 32, 35, 37, 40, and 48 kD that were different from proteins encoded by HSP mRNAs. The induction of HAPs during acute hypoxia and their continued synthesis in chronic hypoxia suggest that HAPs may be important in the maintenance of endothelial cell integrity under conditions of decreased ambient oxygen.