The hsp 70 gene of Drosophila contains three domains to which a heat shock gene specific transcription factor (HSTF) binds. In addition to the previously described 55 bp binding domain proximal to the TAT homology, there are two 25 bp binding sites farther upstream. Footprinting studies with 5' and 3' deletion mutations show two contiguous HSTF binding sites of different intrinsic affinities within the 55 bp binding domain. Determinations made with an agarose-acrylamide gel assay suggest that the HSTF possesses a 12.5-fold higher intrinsic affinity for the site closest to the TATA homology than for the more distal site. Binding of HSTF to the distal site thus appears cooperative, requiring occupancy of the first site. Transcription studies in vitro on the 5' deletions with nuclear extracts and reconstitution experiments show that the TATA proximal site alone, is insufficient for maximal transcriptional activation of the hsp 70 gene.