Human prostate-specific transglutaminase (hTG_P) is a cross-linking enzyme, the physiologic function of which has not been established unequivocally yet. To gain insight into its distribution, we raised antisera against hTG_P. By using Western blotting analysis, we found that these antisera specifically recognize a 77-kDa protein in prostatic fluids, seminal plasmas, and prostatic tissues. The concentrations of hTG_P in these fluids and tissues were found to be highly variable among individuals. Immunohistochemical examination of several formalin-fixed paraffin-embedded human tissues revealed an exclusive expression in the prostate. The histologic localization and distribution of hTG_P within the prostate was assessed by studying multiple sections from tumor-containing prostatectomy specimens and needle biopsies. hTG_P expression was entirely restricted to luminal epithelial cells. No basal epithelial cells or stromal cells were stained. Within the prostate, large areas without any hTG_P-positive cells were seen. Immunopositive cells were present either in a scattered pattern or concentrated in single or multiple glands in which all luminal epithelial cells expressed hTG_P. The latter staining pattern occurred frequently, but not exclusively, in the peripheral zone, whereas scattered expression was most often observed in the transitional zone. Expression of the hTG_P protein could occasionally be observed in high-grade prostatic intraepithelial neoplasia, but was not detected in prostate carcinoma cells. The expression pattern as observed for hTG_P has not been found thus far for any other prostate-specific marker.