TFIIIB, the initiation factor for transcription by RNA polymerase III (pol III) is, in yeast, composed of three subunits: TBP, TFIIIB70/Brf1 and TFIIIB90. To determine the extent to which each of these subunits is limiting for pol III transcription, the effect of overexpressing each subunit was assessed on the expression of wild-type and promoter mutant pol III genes both in vivo and in vitro . In vivo , we find that the synthesis of wild-type pol III genes is not limited to a significant extent by the level of any TFIIIB subunit. There is, however, a two-fold increase in the synthesis of the promoter mutant gene, sup9-e A19-supS1 , in strains overexpressing TFIIIB70. The findings suggest that overexpression of TFIIIB70has a differential effect on the expression of pol III genes with strong versus weak promoters. In vitro transcription assays support this conclusion and reveal an inverse correlation between the transcriptional response to TFIIIB70overexpression and promoter strength. The individual TFIIIB subunits are nuclear by immunofluorescence and are calculated to have nuclear concentrations in the low micromolar range. In comparison, the factors are diluted 100-fold or more in whole cell extracts. This dilution accounts for the generally limiting nature of TFIIIB70in pol III gene transcription in vitro.