We used in silico (electronic database) subtraction to identify ESTs that are preferentially expressed in the adult mouse germ cells. During our analysis, we identified Obox1 and Obox2 transcripts as preferentially expressed in the mouse unfertilized egg libraries. Obox1 and Obox2 transcripts encode homeodomain proteins of 204 amino acids that share 97% identity with each other. Further characterization of mouse BACs encoding Obox1 and Obox2, as well as available BAC and EST sequences in GenBank, identified four closely related genes: Obox3, Obox4, Obox5, and Obox6. Northern blot analyses and RT-PCR from 10 different adult mouse tissues showed that the six Obox family transcripts are preferentially expressed in the gonads. In situ hybridization detected Obox1 and Obox6 transcripts exclusively in oocytes as early as one-layer follicles and throughout folliculogenesis. Obox1, Obox2, Obox3, Obox4, Obox5, and Obox6 map to proximal chromosome 7 in the mouse. The Obox1 and Obox2 genomic structures revealed the presence of six exons each. The Obox genes represent a new family of tissue-specific homeobox genes preferentially expressed in gonads.