We have analyzed 41 deletion, linker scan, and substitution mutants of the human HSP70 gene promoter for activation by the adenovirus E1a region. No natural element of the HSP70 gene promoter was required for activation. To investigate specific interactions between E1a and transcription factors, a set of 24 promoters containing all possible combinations of eight different upstream or TATA motifs was investigated for E1a stimulation. E1a transactivated the promoter regardless of the particular TATA motif present. Furthermore, there was no dramatic correlation between any upstream motif and activation by E1a. These data suggest that E1a does not stimulate transcription via an interaction with any specific transcription factor but instead suggest that E1a interacts via the general transcription machinery.