The male-enhanced antigen-1 gene (Mea1) was originally isolated from a murine testicular cDNA library using anti-H-Y antigen antisera and was assigned to chromosome 17. On analysis of its structure and expression, we found that the Mea1 genomic sequence is flanked by two other genes: Ppp2r5d present in its 3'-terminus in a tail-to-tail orientation and a novel gene called Peas in its 5'-terminus in a head-to-head orientation. The coding sequences of the two genes embedded in the Mea1 sequence are located on the opposite DNA strands of Mea1. Cap-site analysis of Mea1 revealed that it is transcribed from at least seven sites. Most splice variants of Mea1 were abundantly expressed in the testis; the d-type was weakly expressed in the other tissues. AP-2-binding motifs were detected in the transcription-initiation sites. In situ hybridization and immunohistochemical studies revealed Mea1 expression in pachytene spermatocytes. This expression was most prominent in spermatids and residual bodies. The Mea1 protein was also localized in the cytoplasm of elongated spermatids and residual bodies. Localization of the Mea1 suggests that it may function in the very late stages of spermiogenesis. The possibility that Mea1 is one of the serologically detectable male antigens is discussed.