The heat shock response, resulting in the production of heat shock proteins or molecular chaperones, is triggered by elevated temperature and a variety of other stressors. Its master regulator is heat shock transcription factor 1 (HSF1). Heat shock factors generally exist in multiple isoforms. The two known isoforms of HSF1 differ in the inclusion (HSF1α) or exclusion (HSF1β) of exon 11. While there are some data concerning the differential expression patterns and transcriptional activities of HSF2 isoforms during development, little is known about the distinct properties of the HSF1 isoforms. Here we present evidence for two novel HSF1 isoforms termed HSF1γα and HSF1γβ and we show that the HSF1 isoform ratio differentially regulates heat shock protein gene transcription. Hsf1γ isoforms are expressed in various mouse tissues and are translated into protein. Furthermore, after heat shock, HSF1γ isoforms are exported from the nucleus more rapidly, or degraded more quickly than HSF1α or HSF1β. We also show that each individual HSF1 isoform is sufficient to induce the heat shock response and that expression of combinations of HSF1 isoforms, in particular HSF1α and HSF1β, results in a synergistic enhancement of the transcriptional response. In addition, HSF1γ isoforms potentially suppress the synergistic effect of HSF1α and HSF1β co expression. Collectively, our observations suggest that the expression of HSF1 isoforms in a specific ratio provides an additional layer in the regulation of heat shock protein gene transcription.