The homeobox family of proteins are transcription factors are known to be important during the differentiation of a variety of mammalian tissues, however, expression of the genes encoding homeobox proteins during adipogenesis or in adipose tissue has not been described. To investigate whether members of the homeobox gene family are expressed and regulated during adipocyte differentiation, RNA was isolated from 3T3-L1 preadipocyte cells during the hormonal induced differentiation of this cell line into adipocytes. A reverse transcriptase-polymerase chain reaction strategy using degenerate oligonucleotide primers complementary to the highly conserved homeodomain resulted in the identification of 10 different homeobox genes expressed during 3T3-L1 adipogenesis. One of the clones appears to be unique and 9 of the clones represented known members of the homeobox gene family. Examination of the relative mRNA levels encoding these proteins by ribonuclease protection assay during adipocyte differentiation revealed that 3 members, Hox a4, Hox a7, and Hox d4, are regulated as a function of adipocyte development. Further examination of RNA isolated from murine retroperitoneal adipose tissue revealed that these three regulated homeobox mRNAs are expressed in vivo. Combined, these results suggest that members of the homeobox gene family may serve an important role during the differentiation of adipocytes.