The zinc-finger-Y (ZFY) gene is a candidate for the testis-determining-factor gene (TDF) on the human Y chromosome and is postulated to initiate testis differentiation during embryogenesis. However, the present study indicates that the ZFY gene and its X homologue (ZFX) are differentially expressed in adult tissues. A human testis-specific ZFY cDNA was isolated and completely sequenced. The corresponding ZFY transcript encodes a protein that has 801 amino acids and a calculated molecular weight of 90.6 kD. Expression analysis demonstrated that ZFY is transcribed primarily as 3- and 5.7-kb mRNA in testis and somatic cells, respectively. In contrast, the ZFX gene is expressed as a 5-kb transcript in the testis and as 6.7- and 8-kb transcripts in both ovarian and somatic tissues. With sets of gene-specific oligonucleotides, the origin and relative amount of the respective transcripts can be demonstrated in both Northern hybridization and reverse transcriptase-polymerase chain reaction analysis. Significantly, the 3-kb ZFY transcript was also detected in other mammalian adult testes. The testis-specific transcription of the ZFY gene hence suggests that it serves a conserved function in this organ.