We showed previously that HeLa cell nuclear extracts contain two forms of transcription factor IIIC (TFIIIC) that formed chromatographically distinct TFIIIC-promoter complexes (Hoeffler, W. K., Kovelman, R., and Roeder, R. G. (1988) Cell 53, 907-920). One of these forms, the upper-band form, correlated with TFIIIC transcriptional activity, whereas the lower-band form bound to the VA1 promoter but supported little or no transcriptional activity. Using both transcription and DNA-binding assays, we have now purified both the upper-band form and the lower-band form of TFIIIC to near-homogeneity. The upper-band form is composed of five polypeptides with estimated sizes of 220, 110, 102, 90, and 63 kDa. The largest of these polypeptides can be cross-linked to the VA1 promoter. The lower-band form has a polypeptide structure similar to that of the upper-band form except for the absence or modification of the 110-kDa subunit. Direct assays show that the lower-band form is indeed transcriptionally inactive at all stages of purification, even when assayed with an unfractionated, heat-treated nuclear extract as a complementation system. This inactivity does not result from altered DNA-binding properties; instead, we suggest that the alteration of one of the subunits of TFIIIC renders it unable to interact productively with a downstream component of the transcription complex.