Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional enzyme best known to many investigators as a 'housekeeping' gene used as a loading control on northern blots. Prior studies, however, have shown that GAPDH RNA levels vary greatly among different prostate cancer cell lines. We undertook this study to determine the level of GAPDH gene expression within primary human prostate tumors and to determine the validity of using GAPDH as a loading control for northern analysis of human prostate cancer specimens and normal human organs. We found that GAPDH expression was significantly higher in specimens from patients with pathological stage C and D prostate cancer than those with stage B disease. Within the human prostate cancer cell lines TSUPr1, DU145, LNCaP and PC-3 and a normal neonatal prostate epithelial cell line FNC 267beta1, LNCaP cells expressed the highest level of GAPDH but all cell lines, including the normal neonate prostate cell line had very strong GAPDH gene expression. GAPDH RNA levels were highly variable among 16 normal human adult organs and four human fetal organs examined. We conclude that GAPDH RNA levels in human prostate tumors correlate with pathologic stage and that GAPDH should not be used as a loading control in northern blot experiments. Other controls, such as beta-actin or 28s ribosomal RNA, would be more suitable for such purposes.