Six variants of P12, a Kazal-type trypsin inhibitor in the secretion of male mouse accessory sexual glands, were made using single-site mutations including R19L, Y21V, D22G, R43G, K44S, and R45T, based on one-letter-code mutation of amino acids. The other two variants, Nd10 and Cd8, were made using the deletion of 10 and 8 residues from the N- and C-terminals, respectively. Their CD profiles revealed maintenance of the P12 conformation in the seven variants, excluding Cd8, which became unfolded. Only R19L entirely lost the ability while the other variants were as strong as P12 in inhibiting the trypsin digestion of N-benzoyl-Phe-Val-Arg 7-amido-4-methylcoumarin. The immunocytochemical results demonstrated that D22G and Cd8 failed to bind to sperm, Y21V very weakly did so, and the other variants retained their sperm-binding abilities. Concomitantly, the immunocytochemical stainability of each ligand was parallel to its inhibitory effect on ¹²⁵I-P12-sperm binding, and a synthetic oligopeptide corresponding to residues 18–24 of P12 was able to inhibit P12-sperm binding. The data together concluded that R¹⁹ was essential for protease inhibition and D²² and/or Y²¹ mainly being responsible for the binding of P12 to sperm. The steric arrangement of R¹⁹, Y²¹, and D²² on the tertiary structure of P12 is discussed.