Gpbox is a paired-like homeobox gene that colocalizes with two other members of the family, PsxI and Pem, on the proximal portion of the mouse X chromosome. Gpbox is expressed in the extraembryonic placenta and within the germ cells of the embryonic gonad. Beginning with the onset of sexual dimorphism (embryonic day [E]11.5 to 12.5), GPBOX transcripts accumulate faster in female than in male germ cells but disappear later in embryogenesis (E16) and have not been reported in adult tissues. To investigate the function of Gpbox, mouse cell lines lacking GPBOX were established using targeted mutagenesis in embryonic stem cells. Both homozygous Gpbox null female and hemizygous Gpbox null male mice were fertile and reproduced normally. Additionally, the development of male and female gonads in the null background was indistinguishable from that observed in normal littermates. The lack of an obvious phenotype raises the possibility that another member of this homeobox gene family provides the absent Gpbox function.