ABSTRACT: A specific and sensitive “sandwich”-type radiolabeled antiglobulin assay (RAA) using monoclonal anti-human C5b-9 neoantigen and polyclonal anti-human C5b-9 was used to evaluate the presence of the in vivo product of human complement (C) activation (SC5b-9) in the seminal plasma (SP) of 19 fertile and 61 infertile men. SP SC5b-9 was detectable in 7 (8.7%; 1 fertile and 6 infertile men) of the 80 men with a range of 10 to 175 ng/ml. Levels of SP SC5b-9 in other men were below the limit of detection (less than 10 ng/ml). Of the 33 infertile men with sperm-associated immunoglobulin (Ig) G and/or IgA, 27 (82%) had undetectable levels of SP SC5b-9 immunoreactivity. There was no correlation between the SP SC5b-9 levels and the degree of sperm-associated IgG (r = 0.086) or IgA (r = 0.23) activity. However, significant deposition of sperm-bound C5b-9 due to autologous C activation was demonstrated by flow cytometry of donor sperm treated with sera from autoimmune men with ASA in their sera and on their sperm. These findings suggest that sperm-bound Ig cannot activate C in SP.