In previous studies, we have shown that the PCF1-1 mutation of Saccharomyces cerevisiae suppresses the negative effect of a tRNA gene A block promoter mutation in vivo and increases the transcription of a variety of RNA polymerase III genes in vitro. Here, we report that PCF1 encodes the second largest subunit of transcription factor IIIC (TFIIIC) and that the PCF1-1 mutation causes an amino acid substitution in a novel protein structural motif, a tetratricopeptide repeat, in this subunit. In agreement with the nature of the mutation, in vitro transcription studies with crude extracts indicate that PCF1-1 facilitates the rate-limiting step in transcription, namely, the recruitment of TFIIIB to the template. Additionally, biochemical fractionation of wild-type and mutant cell extracts shows that PCF1-1 increases the amount of the 70-kDa TFIIIB subunit detectable by Western (immunoblot) analysis in purified TFIIIB fractions and the transcription activity of a TFIIIB" fraction containing the 90-kDa subunit of this factor. We suggest that the effect of PCF1-1 on TFIIIB activity in vitro is a consequence of its increased rate of recruitment in vivo.