ABSTRACT: Semenogelin I and II (Sglt Sgll) are two separate gene products of chromosome 20 with extensive (∼80%) identity in primary structure. They are mainly responsible for immediate gel formation of freshly ejaculated semen. Degradation of Sgl and Sgll is due to the proteolytic action of prostate-specific antigen (PSA); it results within 5–15 minutes in liquefaction of semen and release of progressively motile spermatozoa. By means of cDNA cloning and Northern blots, Sgl and Sgll transcripts have previously been shown to be abundant in human seminal vesicles, but Sgll alone is suggested to be expressed at low levels in the epididymis. To characterize the expression and tissue distribution of Sgl and Sgll in greater detail, we produced monoclonal immunoglobulin Gs (IgGs) for immunocytochemistry (ICC) and specific [35S]-, digoxigenin-, or alkaline phosphatase-labeled 30-mer antisense probes to Sgl and Sgll for in situ hybridization (ISH). Immunocytochemical staining for both Sgl and Sgll, and ISH detection of both Sgl and Sgll transcripts, were demonstrated in the cytoplasm of seminal vesicle epithelium. ISH showed Sgll alone to be expressed in the epithelium of the epididymal cauda. Neither ICC nor ISH yielded any evidence of Sgl or Sgll expression in caput or corpus epithelium or in any stromal cells of the epididymis. Consistent with our previous findings using polyclonal IgG, monoclonal anti-Sgl/Sgll IgGs identified epitopes on the posterior head, midpiece, and tail of ejaculated spermatozoa. Spermatozoa in the epididymal cauda were also immunoreactive, but those in the caput or corpus region of the epididymis as well as those in the testis were negative. As shown by ICC, neither Sgl nor Sgll were expressed in the testis, the prostate, the female genital tract, or other normal human tissue specimens. Although the significance of Sg attachment to epididymal and ejaculated spermatozoa remains to be established, monoclonal anti-Sg IgG might prove useful in establishing the origin of seminal vesicle tissue components in prostate core biopsies or other biopsy specimens.