Human sperm-free seminal plasma (HSP) contains inhibitors (I) of the seminal plasma histone kinase activity (HK). One I is dialyzable and the other I is nondialyzable and precipitable by dialysis of HSP against a hypotonic buffer. When the nondialyzable, precipitable I fraction is resolubilized, it inhibits HK in a concentration-dependent manner. Sephadex G-25 column chromatography of whole HSP resolves I in both the void (Vo) and inclusion (Vi) volumes. Rechromatography of the VoI resolves I solely in the Vo. These and other data suggest that the ViI does not originate from the VoI, and that both I activities represent separate molecular entities. VoI was further characterized and found to be heat labile, trypsin and neuraminidase insensitive, and alpha-chymotrypsin sensitive. VoI is not soluble in CHCl3 or CHCl3:CH3OH (2:1) and is not adsorbed by charcoal. Chromatography of VoI on Sephadex G-100 yields a broad peak of I that migrates just past the Vo. VoI has no detectable cyclic AMP (cAMP) binding activity and VoI activity is not affected by coincubation of VoI and HK with cAMP. VoI also does not bind to zinc-chelate or phenothiazine affinity columns. These data suggest that VoI is protein in nature with properties distinct from the class of previously described protein kinase inhibitors. Although the identity of VoI is not known, it does not appear to be the regulatory subunit of a cAMP-dependent protein kinase, calsemin or a zinc binding protein.