Human transcription factor hTFIIIB is necessary to initiate transcription correctly from all RNA polymerase III (pol III) genes which are governed by structurally different promoters, and it is unclear whether hTFIIIB complexes, required for intragenic or 5'-located pol III promoters, are composed of unique or different components. We show here that two different forms of hTFIIIB can be separated physically by ion exchange chromatography. hTFIIIB-alpha shows strong preference for transcription of the U6 over the VAI gene and does not contain TATA binding protein (TBP). After SDS-PAGE and renaturation of proteins, the transcriptional activity of hTFIIIB-alpha can be reconstituted by fractions corresponding to a mean M(r) of 25, 60 and 90 kDa. Upon gradient centrifugation or gel filtration, the activity of hTFIIIB-alpha is associated with an M(r) of 60 +/- 10 kDa, indicating that the components of the complex tend to dissociate. In contrast, hTFIIIB-beta is predominantly active on intragenic pol III promoters. It reveals an M(r) of 300 +/- 30 kDa upon gel filtration and, besides TBP, it contains several associated factors (TAFs). Two of these proteins reveal an M(r) of 60 kDa and 90 kDa, and it is conceivable that they are related to polypeptides of similar mass functionally identified in hTFIIIB-alpha. These protein are probably required for the recruitment of pol III to the initiation site at 5'-located and intragenic promoters.