Methods are described for the partial purification of the Saccharomyces cerevisiae class III gene transcription factor TFIIIB from yeast whole cell extracts. A major component (30% of the total protein) of our most highly purified TFIIIB preparation was a polypeptide with an apparent Mr = 60,000 when analyzed by denaturing polyacrylamide gel electrophoresis. This protein was purified by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and polyclonal antibodies were raised against it. Using immunological methods it was shown that TFIIIB transcription factor activity was associated with this purified polypeptide. Furthermore, the polyclonal sera raised against the yeast TFIIIB polypeptide was also shown to specifically neutralize the activity of the human TFIIIB equivalent when it was added to a human KB cell S-100 in vitro transcription system.