Various major leukocyte subpopulations (granulocytes, monocytes/macrophages, B lymphocytes, helper and suppressor/cytotoxic T lymphocytes) were detectable in human semen by applying monoclonal antibodies (MAbs) specific for pan-leukocyte and leukocyte subpopulation markers. MAb-labeled cells in smears of washed human semen were visualized by a standard streptavidin-biotin immunoperoxidase method. This approach was validated by testing the MAb panel against sections of human testis and epididymis to rule out cross-reactivity with immature germ cells or other reproductive tract cells, and against peripheral blood leukocytes that had been incubated in human semen to ascertain leukocyte marker stability in seminal plasma. When the immunoperoxidase technique was applied to semen smears from 17 proven fertile men and 51 randomly selected infertility patients, the authors found highly varying total leukocyte numbers, ranging from 8970 to 20,520,000 per ejaculate among fertile men (median, 170,000), and from 43,120 to 104,580,000 among infertility patients (median, 1,035,000). Higher means and medians of granulocytes, monocytes/macrophages, and lymphocyte subsets were seen in the infertility group. These results warrant further investigation on the diagnostic utility of semen leukocyte profiles and the possible role of seminal leukocytes in male infertility.