Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional enzyme overexpressed in many tumors and induced by hypoxia in normal and malignant cells. The degree to which hypoxia transcriptionally activates GAPDH is cell type specific. The GAPDH promoter region contains a hypoxia responsive element (HRE) consisting of a hypoxia inducible factor-1 (HIF-1) consensus binding site plus adjacent sequence [Graven et al. (1999) Biochim. Biophys. Acta 1447, 208-218]. Using transient transfection experiments with the GAPDH promoter region linked to a luciferase reporter gene, we found that GAPDH was transcriptionally activated by hypoxia in each of three human prostate cancer cell lines tested, with the greatest level of induction in the most differentiated cell line. Using sequence analysis of the GAPDH promoter region, we identified a novel HRE distinct from the previously characterized one that consists of two consensus HIF-1 sites arranged as inverted repeats separated by 5 bp. Hypoxia transcriptionally activated a promoter construct in which the previously characterized HRE was mutated and the novel HRE remained intact. Heterologous promoter constructs containing only one or two copies of the novel HRE plus a minimal promoter consisting of a TATA box drove hypoxia inducible expression of the luciferase reporter gene in transient transfection assays. Mutation of HIF-1 sites within the novel HRE resulted in complete loss of function.