The 5S gene-specific transcription factor, TFIIIA, was purified approximately 35,000-fold from HeLa cell extracts using a combination of conventional and affinity chromatographic methods. A single polypeptide of apparent molecular mass 42-kDa cofractionates with both 5S DNA binding and 5S transcription activities, and was conclusively identified as human TFIIIA by its ability to direct specific 5S gene transcription in vitro following elution and renaturation from SDS-polyacrylamide gels. The DNase I protection pattern of the purified human factor on a human 5S gene is similar to the pattern previously observed with Xenopus TFIIIA. A Xenopus 5S gene, but not a yeast 5S gene, is able to effectively compete for binding of the human factor. In addition, we report a previously undetected immunological cross-reactivity between human and Xenopus TFIIIA. hTFIIIA is recognized specifically both by polyclonal antisera raised against Xenopus laevis TFIIIA as well as by a monoclonal antibody generated against the amphibian protein. These observations indicate that human TFIIIA is structurally related to the Xenopus oocyte factor and that the previous inability to detect human TFIIIA by immunological methods is due primarily to the low abundance of this factor in HeLa cell extracts.