Human TFIIIC1, a basal transcription factor essentially required for expression of all pol III genes, exerts its function without primarily binding to DNA. We report here the purification of a termination site binding activity (TBA) which was initially described to be contained in fractions designated as TFIIIC0. TBA specifically and strongly binds to the termination region of pol III genes with internal promoters and can be completely separated from TFIIIC1and a TFIIIC1related activity (TFIIIC1-like), proving that DNA-binding of TBA is independent of these latter activites. Although TBA is not essentially required for, it strongly stimulates pol III transcription from intragenic promoters. This stimulation strictly depends on the presence of TFIIIC1and is not observed in conjunction with TFIIIC1-like. We further present the identification of a novel activity, TFIIIU, which is also contained in crude fractions of TFIIIC0. TFIIIU can be separated from TBA by further purification and is essentially involved in transcription of the mammalian U6 gene. TFIIIU cannot be substituted for by any of the established U6 transcription factors and thus represents a novel U6 transcription factor.