Transcription factor IIIC (TFIIIC) is a multisubunit basic TF for RNA polymerase III. It initiates transcription complex assembly on tRNA and related genes by binding to the internal box B promoter element and is also required for transcription of 5S rRNA and other stable nuclear and cytoplasmic RNAs transcribed by polymerase III. In mammalian cells, regulation of TFIIIC activity controls overall polymerase III transcription in response to growth factors and viral infection. Here, we report the cloning and sequencing of a full-length cDNA (and genomic DNA from the transcription initiation region) encoding the box B binding subunit of human TFIIIC, the 243-kDa alpha subunit. Specific antisera raised against the encoded protein super shifts a TFIIIC-box B DNA complex during an electrophoretic mobility shift assay and immunodepletes TFIIIC transcriptional activity from a partially purified TFIIIC fraction, proving that the cDNA encodes a component of TFIIIC. The human protein shows surprisingly little similarity to the box B binding subunit of yeast TFIIIC.