ABSTRACT: Beta-glucuronidase (β-glucuronidase) in human seminal plasma consists of two forms of isozymes (major and minor forms). Antiserum prepared against the purified major form of β-glucuronidase in rabbits was used for studies of immunological specificity and immunohistochemical localization of β-glucuronidase in the human male reproductive tract. By ouchterlony immunodiffusion technique, anti-β-glucuronidase serum showed reaction of identity with human tissue extracts such as liver, spleen, lungs, testis, epididymis, prostate, and seminal vesicles. Though monkey testis and epididymis extracts completely reacted with human seminal β-glucuronidase antiserum, no cross-reaction was observed with mouse and rat tissue extracts. Immunofluorescent studies revealed that β-glucuronidase is localized in the early stages of spermatogenesis in testis, in columnar cells of the epididymis, and in glandular cells of the prostate and seminal vesicles. Consistent absence of fluorescence in the lumens of testis and epididymis as well as negative cross-reaction between antiserum and sperm extracts suggested that major form of β-glucuronidase in human seminal plasma is not immunologically identical with human spermatozoa. It is concluded that major form of β-glucuronidase in seminal plasma is not tissue-specific although it appears to have limited species specificity.