The Saccharomyces cerevisiae HSP70 gene SSA1 has multiple heat shock elements (HSEs). To determine the significance of each of these sequences for expression of SSA1, we analyzed expression from a set of promoters containing point mutations in each of the HSEs, individually and in pairwise combinations. Of the three HSE-like sequences, two (HSE2 and HSE3) were active promoter elements; only one, HSE2, was active under basal growth conditions. Either HSE2 or HSE3 alone was able to drive SSA1 transcription at near-normal rates after heat shock. Both HSE2 and HSE3 were capable of driving basal transcription when placed in the context of the CYC1 promoter. Previous analysis had identified an upstream repressing sequence overlapping HSE2 that repressed basal transcription driven by HSE2. Our analysis showed that basal transcription driven by HSE3 was repressed both by the distant upstream repressing sequence and by closer flanking sequences. The ability to drive basal transcription is not inherent in all natural HSEs, since the HSEs from the heat-inducible SSA3 and SSA4 genes showed no basal activity when placed in the CYC1 vector. Gel mobility shift experiments showed that the same population of heat shock transcription factor molecules bound to HSEs capable of driving basal activity and to HSEs having very low or undetectable basal activity.