The present study characterizes the sperm protein Sp17 in the mouse. Sp17 is a mammalian testis- and sperm-specific protein that has been isolated, sequenced, and characterized from rabbit testis and spermatozoa. In this study, a rabbit Sp17 cDNA probe representing the entire protein coding region was used to screen a mouse testis cDNA library to obtain the mouse Sp17 sequence. The mouse mRNa for Sp17 encodes a 149-amino acid protein with a predicted molecular weight of 17,296. The mouse Sp17 (MSp17) cDNA sequence is 82% identical to the rabbit Sp17 cDNA sequence while the MSp17 protein sequence is 74% identical to the rabbit protein sequence. The presence of native Sp17 in mouse spermatozoa and testis was demonstrated by Western blot analysis, immunoprecipitation, and immunolocalization. After SDS-PAGE, the native Sp17 has an apparent molecular mass of 24 kDa. The sequence of the native Sp17 was confirmed by Western blots of mouse testis and spermatozoa probed with two anti-peptide antibodies--one, anti-G22C, made against amino acids 61-82 in the rabbit sequence (61-83 in the mouse), and a second, anti-K18C, made against amino acids 120-136 in the C-terminal region in the human sequence (118-134 in the mouse sequence). In the absence of proteolytic inhibitors, part of the C-terminal of native MSp17 is cleaved, giving rise to an 18-kDa band. Sp17 is present in spermatocytes and spermatids in the testis. In spermatozoa, Sp17 is not available to bind antibody on the surface of live, acrosome-intact spermatozoa, but it is present on the equatorial surface of live, acrosome-reacted spermatozoa. In fixed spermatozoa, staining is observed along the length of the principal piece, weakly along the midpiece, and over the acrosomal region of the head. When the acrosome reaction begins, acrosomal staining is seen throughout the equatorial region of the acrosome. Using mimotope analysis, this study has also demonstrated that native Sp17 is a sperm autoantigen and that recombinant mouse Sp17 is immunogenic in males with a highly restricted linear epitope.