Regulatory factor X (RFX) is a transcription factor that binds the conserved X1 box of MHC class II promoters and is essential for transcription of class II genes. The subunit structure of the native RFX complex was examined by coimmunoprecipitation using polyclonal antisera to the 75-kDa subunit of RFX, RFX5. Two polypeptides with apparent masses of 41 and 36 kDa coimmunoprecipitated with RFX5 and appear to be subunits of the native RFX complex. Metabolic labeling of wild-type and mutant B cells with [32P]orthophosphate demonstrated that each of the RFX subunits was phosphorylated in vivo and that the phosphorylation of the RFX subunits was independent of the essential MHC class II regulatory factor, CIITA. The trimeric RFX complex was also present in fibroblast cells with or without IFN-gamma treatment. Both the p41 and p36 subunits were absent in immunoprecipitations of RFX5 from lysates of independently established B cell lines from bare lymphocyte syndrome complementation groups B and D. Together, these results suggest that RFX complex assembly is required for class II expression and that the mutations in bare lymphocyte syndrome complementation groups B and D result in an inability to assemble the RFX complex.