Serum was obtained from an infertile woman having antibodies with sperm agglutinating activity. The antibodies interacted with a human sperm membrane protein (hSMP-1) with an estimated Mr of 55 kD. The gene (HSD-1) coding hSMP-1 was isolated from a human testis cDNA expression library and assigned the accession number U12978. The cDNA was conjugated to a prokaryotic expression vector to construct the recombinant vector, pRSET-HSD-I, which was expressed in Escherichia coli. The recombinant hSMP-1 was isolated and used to immunize rabbits to raise polyclonal antibodies. Usingan immunocytochemical technique, hSMP-1 protein was immunolocalized in germ cells of human testis at all stages of spermatogenesis. mRNAs were prepared from 16 different human tissues and analyzed by Northern blot using HSD-1 as probe. A positive reaction was elicited only with testis mRNA. The present findings suggest that the expression of hSMP-1 gene is testis-specific and occurs during the early stages of germ cell differentiation. In a comparative study, the location of the hSMP-I protein in sperm and in germ cells of the seminiferous tubules of rats was determined. The target antigen was immunolocated on the head and tail of rat sperm and in late spermatids and spermatozoa of rat testis. These results suggest that, in the rat, the HSD-1 gene is expressed during spermiogenesis.