The class III gene transcription factor termed TFIIIC has been extensively purified from Saccharomyces cerevisiae. Three polypeptides of 138, 131, and 95 kDa consistently copurified with TFIIIC transcription factor activity. These polypeptides were present in approximately equimolar quantities in all TFIIIC preparations. To determine which, if any, of these polypeptides were involved in TFIIIC activity, rabbit polyclonal antibodies were generated against each of these three polypeptides purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunoblot analyses showed that each of the three antiserum preparations reacted uniquely with the respective polypeptide to which they had been elicited. This lack of cross-reactivity by any of the antiserum preparations suggested that these three polypeptides represented distinct unrelated gene products. Each of the three specific antiserum preparations decreased the mobility of TFIIIC-tDNA complexes in a DNA mobility shift assay. More importantly, all three antiserum preparations directly inhibited the transcription factor activity of TFIIIC. In addition, all three antiserum preparations depleted a solution of TFIIIC transcription factor activity. These results indicated that each of these three polypeptides of Mr = 138,000, 131,000, and 95,000 was a distinct and necessary component of yeast TFIIIC. Immunoblot analyses of immunoaffinity-purified TFIIIC fractions indicated that each of the three antiserum preparations alone could deplete the solution of all three polypeptides. These results suggested that these three polypeptides were tightly associated with one another in solution.