A monoclonal antibody was raised to a fox sperm protein (FSA-1) which was found to be localized to the inner acrosomal compartment of sperm fixed in methanol. Western blots of testicular germ cell membrane extracts probed with this antibody identified a major protein band with a molecular weight of 36,000. Immunofluorescent studies on fox testis sections showed that the antigen is expressed on round and elongating spermatids on a crescent-shaped structure, which probably represents the developing acrosome. An antibody specific for FSA-1 was used to screen a fox testis cDNA library for its cognate gene. An 875-bp cDNA clone was isolated and sequenced revealing an open reading frame. Searches of the GenBank and EMBL databases with the nucleic acid sequence revealed significant homology (86%) of FSA-1 with 406 bases of an unidentified RNA transcript from human fetal brain (EST02625). Northern blot analysis of fox testis RNA samples identified an RNA transcript of approximately 0.9 kb during the months when spermatogenesis is active. Zoo Northern blots (at high stringency) reveal an RNA transcript of a similar size present in testis RNA from dogs and mice. Zoo Southern analysis (high stringency) reveal genomic sequences present in dogs, mice, cattle and sheep. At present, the function of the FSA-1 gene product remains unknown, but it may play a role as a structural protein component of the acrosome.