UDP glucuronosyltransferases (UGT) are expressed in a wide range of tissues in which their levels of expression and distribution are dependent on cell-type specific regulatory mechanisms. The presence of a hepatocyte nuclear factor (HNF) 1 binding site in the proximal promoters of several UGT2B genes has been shown to contribute to their expression in liver cells and possibly other HNF1-containing cell types. In some of these UGT2B genes, a putative pre-B cell homeobox (Pbx) transcription factor binding site is found directly adjacent to the functional HNF1 site. To determine whether this putative Pbx site contributes to the regulation of UGT2B expression, we chose the UGT2B17 gene and investigated the capacity of its Pbx site to bind specific transcription factors and alter promoter activity. The UGT2B17 Pbx site matches a consensus Pbx site known to bind members of the Pbx, Hox, Meis, and Prep1 families of homeodomain-containing proteins and has previously been shown to bind nuclear proteins in DNaseI footprint assays. In this study, we used gel shift and functional assays to show that a Pbx2-Prep1 heterodimer can bind to the UGT2B17 Pbx site and interfere with the binding of HNF1alpha to its site adjacent to the Pbx site. This interaction of Pbx2-Prep1 and HNF1alpha results in down-regulation of HNF1alpha-mediated activation of the UGT2B17 promoter. Modulation of transcription by restricting the binding of transcriptional effectors to their target site is a novel role for Pbx2-Prep1 complexes.