Mammalian alphaB-crystallin is highly expressed both in lens epithelium and lens fibers. In contrast, gammaF-crystallin is highly expressed in the lens fiber cells. Crystallin gene expression in lens is regulated at the level of transcription by a sparse number of specific DNA-binding transcription factors. Here, we report studies on transcriptional regulation of mouse alphaB- and gammaF-crystallin promoters by specific combinations of Pax6/Pax6(5a), large Mafs (MafA, MafB, c-Maf, and NRL), Sox1, Sox2, Six3, and RARbeta/RXRbeta. Two sets of these factors, co-expressed both in lens epithelium and in lens fibers, were tested in co-transfection assays using cultured lens and non-lens cells. Regulation of alphaB-crystallin was studied in the presence of lens epithelial-factors Pax6, MafB, and RARbeta/RXRbeta, and lens fiber-factors Pax6, MafA, c-Maf, and NRL. Pax6 proteins activated the alphaB-crystallin promoter (-162 to +45) with any combination of Mafs. Addition of RARbeta/RXRbeta further increased its promoter activity. Gel shift assays using lens nuclear extracts demonstrated interactions of Pax6, Maf, and retinoic acid nuclear receptor proteins with two lens-specific regions, the distal LSR1 (-147/-118) and proximal LSR2 (-78/-40), of the alphaB-crystallin promoter. In contrast, Pax6 proteins acted as repressors of gammaF-crystallin promoter activity elicited by a combination of large Mafs, Sox, and RARbeta/RXRbeta proteins in transiently transfected lens and non-lens cells. The results show that Pax6 conversely regulates these two lens crystallin promoters. We propose that the opposite roles of Pax6 in crystallin gene regulation are results of different promoter architectures of the alphaB- and gammaF-crystallin genes, developmentally regulated association of transcription factors with the corresponding cis-regulatory sites, and specific recruitment of transcriptional co-activators and co-repressors by Pax6.