MHC class II deficiency found in bare lymphocyte syndrome patients results from the absence or dysfunction of MHC class II transcriptional regulators, such as regulatory factor X (RFX) and class II transactivator (CIITA). Understanding the roles of these factors has been greatly facilitated by the study of genetic defects in cell lines of bare lymphocyte syndrome patients, as well as in cell lines that have been generated by chemical mutagenesis in vitro. The latter group includes MHC class II-deficient lines that are no longer responsive to induction by IFN-gamma. Here, we show that the defect in G1B, one such cell line, is attributed to the lack of functional RFX5, the largest subunit of RFX. The RFX5 gene isolated from G1B cells contains two separate single-base pair mutations. One alteration does not exhibit a phenotype, whereas a leucine-to-histidine mutation eliminates DNA-binding and transactivating functions. This mutation lies outside of previously defined functional domains of RFX5 but within an unusual, leucine-rich region (62-LYLYLQL-68). To further investigate the significance of the leucine-rich region, we targeted all neighboring leucine residues for mutagenesis. These mutants were also unable to transactivate a MHC class II reporter gene, confirming that these leucine residues play an essential role in RFX activity and characterize a novel leucine-rich motif.