The PEPP genes are a recently described subfamily of mouse homeobox genes preferentially expressed in reproductive tissues. Pem, the founding member of the PEPP subfamily, has undergone rapid divergence due to positive selection, rendering the identification of its human orthologue difficult. Here we report the isolation and characterization of two human homeobox genes, hPEPP1 and hPEPP2, that are related to Pem and other PEPP family members. We identified these human genes based on their location in Xq24, which is syntenic to the mouse X-chromosome region containing three PEPP genes: Pem, Psx-1, and Psx-2. We found that hPEPP1 and hPEPP2 are selectively expressed in the testis, where the mouse and rat Pem genes are also expressed. However, unlike all mouse PEPP genes, hPEPP1 and hPEPP2 were not expressed in placenta, which suggests the possibility that the regulation of PEPP genes has significantly changed since the split between hominids and rodents. Although hPEPP1 exhibits highly selective expression in normal tissues, it is aberrantly expressed in tumor cell lines from several different organs, analogous to the expression pattern of mouse and rat Pem but not mouse Psx-1 or Psx-2. We conclude that we identified two human homeobox genes from the PEPP subfamily that are good candidates to encode transcription factors that regulate downstream genes and biological events in the human testis.