The HOX11 homeobox gene was first identified through studies of the t(7;10) and t(10;14) chromosomal translocations of acute T-cell leukemia. In addition, analysis of Hox11^/ mice has demonstrated a critical role for this gene in murine spleen development. A possible mode of in vivo function for the HOX11 protein in these two situations is regulation of target genes following DNA binding via the homeodomain, but little is known about how HOX11 regulates transcription in vivo. By performing transcriptional studies in yeast and mammalian one-hybrid systems, a modular transcriptional transactivation region at the NH₂ terminus of HOX11 has been functionally dissected from other parts of the protein. This NH₂-terminal region includes the previously identified short conserved Hep motif, which itself activates transcription in one-hybrid assays. The importance of the NH₂-terminal region for the function of HOX11 in vivo was assayed by activating a HOX11-dependent gene in NIH 3T3 cells. Activation of this gene was found to be dependent upon an intact homeodomain in HOX11, but maximal activation was obtained only when the NH₂-terminal 50 amino acids of HOX11 was present, showing that this region of HOX11 is important for in vivo transcriptional control of a chromosomal target gene.