An octamer-binding transcription factor, OTF-1, which stimulates transcription of a human histone H2b gene, has been purified from HeLa nuclear extracts. This purification was achieved through the use of DNA affinity chromatography, and the factor was unambiguously identified by renaturation of activity following SDS-polyacrylamide gel electrophoresis. The purified factor retained the ability to efficiently stimulate H2b transcription in a reconstituted in vitro system. This effect was dependent upon an intact octamer element and was observed in the absence of the other H2b promoter elements (except the TATA motif). Furthermore, this activity was not detected in nuclear extracts prepared from cells synchronized in G2, in agreement with the in vivo data showing S-phase-specific utilization of the octamer element. From these data, we conclude that we have purified the bona fide H2b transcriptional regulatory factor.