By employing purified transcription factors and RNA polymerase III (pol III), we generated active pol III transcription complexes on the human 5S rRNA gene. These large complexes were separated by size exclusion chromatography from non- incorporated proteins. In addition, we succeeded in isolating specific intermediate stages of complex formation. Such isolated partial complexes require complementation with the missing activities for full transcription activity. One central finding is that a 5S DNA-TFIIIA-TFIIIC2-TFIIIBbeta complex could be isolated which had been assembled in the absence of the general pol III transcription factor IIIC1. Thus TFIIIC1 is not an assembly factor for other transcription factors. Although pol III has the potential to bind unspecifically to DNA, such polymerase molecules cannot be rendered initiation competent by direct recruitment to a 5S DNA-TFIIIA-TFIIIC2- TFIIIBbeta complex, but this process strictly requires additional TFIIIC1 activity. This clearly demonstrates that in contrast to yeast cells, hTFIIIB(beta), although required, does not suffice for the functional recruitment of polymerase III. These data document that TFIIIC1 is the second transcription factor required for the recruitment of pol III in mammalian cells.