A new proteinase inhibitor (Mr 7500) was isolated to apparent homogeneity from boar spermatozoa by repeated gel filtration on Sephadex G-50 and affinity chromatography on concanavalin-A--Sepharose 4B. The inhibitor strongly inhibits boar acrosin in a competitive 1:1 stoichiometric reaction with Kass = 7 x 10(10)1 mol-1. The inhibitor is a glycoprotein and represents a first member of a new class of proteinase inhibitor with a rather short polypeptide backbone of only 42 amino acid residues and a low cystine content. The basic protein (isoelectric point 9.4) contains a single disulfide loop, which is easily reducible by sodium borohydride. Upon reduction the inhibitory activity is lost, but rapidly regained after air reoxidation of the corresponding half-cystine residues. The reactive site residue was established to be arginine by inhibition with 2,3-butanedione. The inhibitor is rather specific for acrosin and inhibits bovine trypsin only to a limited extent. However, incubation with catalytic amounts of trypsin (or acrosin) at acid pH (pH2-3) rapidly leads to a limited proteolysis at the reactive site with formation of 67% modified (reactive site hydrolysed), but still active inhibitor. The equilibrium constant was established to be Khyd = 2.0.