Heat shock factor 1 (HSF1) is essential for the stress-induced expression of heat shock genes. On exposure to heat shock, HSF1 localizes within seconds to discrete nuclear granules. On recovery from heat shock, HSF1 rapidly dissipates from these stress granules to a diffuse nucleoplasmic distribution, typical of unstressed cells. Subsequent reexposure to heat shock results in the rapid relocalization of HSF1 to the same stress granules with identical kinetics. Although the appearance of HSF1 stress granules corresponds to the hyperphosphorylated, trimeric DNA-binding state of HSF1 and correlates temporally with the inducible transcription of heat shock genes, they are also present in heat-shocked mitotic cells that are devoid of transcription. This finding suggests a role for HSF1 stress granules as a nuclear compartment for the temporal regulation and spatial organization of HSF1 activity and reveals new features of the dynamics of nuclear organization.