Transcription factor IIIB (TFIIIB), which by itself does not bind stably or specifically to DNA, was purified from cytoplasmic extracts of HeLa cells using five different chromatographic steps. This procedure yields one predominant polypeptide which represents 90% of the most highly purified preparation and shows a relative molecular mass of 60,000, when analyzed on sodium dodecyl sulfate-polyacrylamide gels. A similar value was obtained for the native protein by rate zonal centrifugation on glycerol gradients. From these data we conclude that TFIIIB from HeLa cells has a Mr of 60,000 +/- 5,000 and that it functions as a single polypeptide. Highly purified TFIIIB was required and sufficient for the specific transcription of the Xenopus laevis and human tRNA and 5 S RNA genes as well as those for VA RNA when reconstituted with RNA polymerase III and the other appropriate transcription factors.