We characterized the activity of a human hsp70 gene promoter by in vitro transcription. Analysis of 5' deletion and substitution mutants in HeLa nuclear extracts showed that the basal activity of the promoter depends primarily on a CCAAT-box sequence located at -65. A protein factor, CCAAT-box-binding transcription factor (CTF), was isolated from HeLa nuclear extracts and shown to be responsible for stimulation of transcription in a reconstituted in vitro system. DNase I footprinting revealed that CTF interacts with two CCAAT-box elements located at -65 and -147 of the human hsp70 promoter. An additional binding activity, heat shock transcription factor (HSTF), which interacted with the heat shock element, was also identified in HeLa extract fractions. This demonstrates that the promoter of this human hsp70 gene interacts with at least two positive transcriptional activators, CTF, which is required for CCAAT-box-dependent transcription as in other promoters such as those of globin and herpes simplex virus thymidine kinase genes, and HSTF, which is involved in heat inducibility.