The human sperm antigen SP-10 is a testis-specific, intra-acrosomal protein associated with the membranes of the acrosomal vesicle. The molecule has been designated a "primary vaccine candidate" by a World Health Organization (WHO) Taskforce on Contraceptive Vaccines. cDNA cloning and sequencing have indicated that SP-10 is encoded by a 795-base-pair (bp) reading frame that predicts a 265-amino acid protein of 28.3 kd. In this study, we used a 634-bp fragment (bp 68 through 700, amino acids 3 through 222) of the SP-10 sequence to probe, by Southern blotting, EcoRI-digested DNA from 33 mouse/human somatic cell hybrids involving 16 unrelated human cell lines and 4 mouse cell lines. The hybrids were characterized by karyotypic analysis and by mapped enzyme markers. The presence or absence of positive human bands was scored on the blots and the percent of concordance and discordance with a specific human chromosome was determined. The DNA probe for SP-10 showed a concordance of 31 and a discordancy of 0 for human chromosome 11, mapping SP-10 unequivocally to this chromosome. The hybrid XER-7 with the 11/X translocation: 11p12 or 11p11----11qter:: Xq11----Xqter and the hybrid EXR-5CSAZ with the X/11 translocation: Xpter----Xq22::11q13----11qter localized the SP-10 gene to the p12----q13 region. The SP-10 locus has been assigned the gene symbol ACRV1 (acrosomal vesicle protein-1).