Human 5'-nucleotidase (5'-NT, EC 3.1.3.5) is an enzyme that hydrolyzes nucleotides such as AMP or IMP (inosine 5'-monophosphate) into inorganic phosphate and the respective nucleoside. It has been suggested that the enzyme acts as a scavenger of injured cell or membrane components or as a supplier of adenosine. We have purified to homogeneity human 5'-NT, a 69-kDa glycoprotein containing a glycosylphosphatidylinositol anchor, present in human seminal fluid. With use of a polyclonal rabbit antiserum against the protein, a strong immunoreaction was detected in prostatic epithelium, exceeding that in placental syncytiotrophoblast and amnion cells. A slightly less intense immunoreaction was present in some cells of seminal vesicle epithelium and in vesicular intraluminal secretion. In the epididymis, only the apical cell portion and particularly the stereocilia of the epididymal principal cells, as well as clusters of small nonciliated cells in the efferent ductules, were immunoreactive. In the testis, no immunoreactive cells at all were detected, and likewise no clear-cut signal was observed in testicular and epididymal spermatozoa. The immunohistochemical results were coincident with Western blots prepared from homogenates of the respective tissues. Reverse transcription-polymerase chain reaction studies were performed with primers derived from the sequence of human placental ecto 5'-NT. Using human placenta as a reference tissue, positive results were obtained in the epididymis, seminal vesicle, and prostate, but not in the testis. On Northern blots, we determined the size of the mRNA at 2.4 kilobases. The relatively strong expression of 5'-NT in the human male accessory sex glands points to a potential regulatory role of the enzyme during posttesticular modification of the sperm surface.