The promoter of the mouse gene encoding ribosomal protein L30 contains binding sites for four transcription factors; alpha (RFX-1), beta (GABP), gamma and delta (YY-1/NF-E1/UCRBP). The relative contributions of these factors to the strength of the rpL30 promoter in vivo and the degree of synergism among the factors was evaluated by transfection experiments using a series of mutant promoters in which one or more of the binding sites was drastically altered to prevent recognition by its cognate factor. Our results indicated that GABP and RFX-1 are the major determinants of the rpL30 promoter strength, acting synergistically to boost activity more than eightfold over that which occurs in their absence. The contributions of gamma and delta became evident only when the promoter was weakened by eliminating the participation of the other factors. Indeed, as the promoter strength was progressively reduced, the contribution of each individual factor increased, implying that the capacity of the general transcription machinery to be stimulated by these factors is saturable. The activity of the rpL30 promoter was significantly diminished when three pyrimidine residues spanning the start site were converted to purines, indicating that the integrity of the oligopyrimidine tract is also a determinant of the transcriptional efficiency. These studies reveal the hierarchy of importance of four transcription factors that govern the expression of the rpL30 gene. Moreover, they define the graduated levels of promoter activity that would result from deficiencies of these factors in any particular cell type. This information may provide a useful paradigm for understanding the transcriptional regulation of other ubiquitously expressed genes.