The TFIIIB transcription factor is the central component of the RNA polymerase III transcriptional machinery. In yeast, this factor is composed of three essential polypeptides TBP, TFIIIB70 and TFIIIB90, that are sufficient as recombinant proteins, together with TFIIIC, to promote accurate transcription in vitro. Here we show that a partially purified fraction, named B", that contains the TFIIIB90 subunit, displays properties distinct from recombinant TFIIIB90. This fraction contains at least a component that interacts with DNA*TFIIIC complexes, either alone or in combination with TFIIIB90, and increases the resistance of the complexes to heparin treatment. In addition, primer extension and single round transcriptions experiment reveal a different start-site selection pattern directed by B" or rTFIIIB90. In mixing experiments, we show that an activity in B", distinct from TFIIIB90, can promote transcription initiation at the +1 site without affecting the rate of preinitiation complex formation. Our data suggest the existence of at least one new component that participates in preinitiation complex formation and influences start-site selection by RNA polymerase III.