TSPY, the 'testis-specific protein, Y-encoded', is the product of a tandem gene cluster on human proximal Yp. In order to gain insight into the function of this locus, we have analysed (I) the diversity of RNAs transcribed from the cluster, (II) the sequence homology of the deduced TSPY to other proteins, and (iii) its protein properties both in tissue extracts and in tissue sections, using a TSPY-specific antiserum. We have identified a set of distinct TSPY transcripts with diverse exon compositions. We show that TSPY has homology with other human and non-human proteins, including SET and NAP, factors that are suggested to play a role in DNA replication. Protein analysis revealed TSPY to occur mainly in a modified, putatively phosphorylated form. By immunostaining it was detected in distinct subsets of spermatogonia. TSPY was also strongly immunostained in early testicular carcinoma in situ (CIS), while seminomatous tumour cells stained less intensely. The spermatogonial cells of two XY-TFM-females gave a strong immune response. The data presented here point to a phosphorylation-dependent TSPY-function in early spermatogenesis, immediately prior to the spermatogonia-to-spermatocyte transition, and in early testicular tumorigenesis.