Exposure of endothelial cells (EC) to hypoxia results in the increased expression of a distinct set of proteins with molecular masses of 56, 47, 39, 36, and 34 kDa. Their induction appears to be unique to EC and the stress of decreased oxygen tension. To understand the mechanism(s) and significance of the up-regulation of these proteins we have identified the 36-kDa protein by limited amino-terminal amino acid sequencing. The 21-amino acid sequence from the bovine protein exhibited 90.5% identity with the human sequence of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Northern blot analysis showed that the time course and extent of EC GAPDH mRNA up-regulation correlated with the increase in 36-kDa protein synthesis. Nuclear runoff analysis demonstrated that this increase in GAPDH expression is regulated, in part, at the transcriptional level; however, the increase in the rate of transcription did not account for the entire mRNA accumulation, suggesting that GAPDH, like other hypoxia-regulated proteins, is posttranscriptionally regulated. Subcellular fractionation of hypoxic EC showed up-regulation of the 36-kDa protein in the cytoplasmic fraction and, to a lesser extent, in the nuclear fraction. The up-regulation of GAPDH in EC may be related to their relative hypoxia tolerance. Alternatively, the up-regulation of GAPDH in EC during hypoxia may be related to the potential nonglycolytic functions of this enzyme.