Hox genes contain a homeobox encoding a 60-amino acid DNA binding sequence. The Hoxa1 gene (Hox1.6, ERA1) encodes two alternatively spliced mRNAs that encode distinct proteins, one with the homeodomain (Hoxa1-993), and another protein lacking this domain (Hoxa1-399). The functions of Hoxa1-399 are unknown. We detected Hoxa1-993 and Hoxa1-399 by immunoprecipitation using Hoxa1 antibodies. To assess whether Hoxa1-399 functions in cellular differentiation, we analyzed Hoxb1, a Hoxa1 target gene. Hoxa1-993 and its cofactor, Pbx1, bind to the Hoxb1 SOct-R3 promoter to transcriptionally activate a luciferase reporter. Results from F9 stem cells that stably express ectopic Hoxa1-399 (the F9-399 line) show that Hoxa1-399 reduces this transcriptional activation. Gel shift assays demonstrate that Hoxa1-399 reduces Hoxa1-993/Pbx1 binding to the Hoxb1 SOct-R3 region. GST pull-down experiments suggest that Hoxa1-399, Hoxa1-993, and Pbx1 form a trimer. However, the F9-399 line exhibits no differences in RA-induced proliferation arrest or endogenous Hoxb1, Pbx1, Hoxa5, Cyp26a1, GATA4, or Meis mRNA levels when compared to F9 wild-type.