Post-heat shock refolding of luciferase requires chaperones. Expression of a dominant negative HSF1 mutant (dnHSF1), which among other effects depletes cells of HSF1-regulated chaperones, blocked post-heat shock refolding of luciferase targeted to the cytoplasm, nucleus, or peroxisomes, while refolding of endoplasmic reticulum (ER)-targeted luciferase was inhibited by about 50 %. Luciferase refolding in the cytoplasm could be partially restored by expression of HSPA1A and fully by both HSPA1A and DNAJB1. For full refolding of ER luciferase, HSPA1A expression sufficed. Neither nuclear nor peroxisomal refolding was rescued by HSPA1A. A stimulatory effect of DNAJB1 on post-heat shock peroxisomal luciferase refolding was seen in control cells, while refolding in the cytoplasm or nucleus in control cells was inhibited by DNAJB1 expression in the absence of added HSPA1A. HSPB1 also improved refolding of peroxisomal luciferase in control cells, but not in dnHSF1 expressing cells. HSP90, HSPA5, HSPA6, and phosphomevalonate kinase (of which the synthesis is also downregulated by dnHSF1) had no effect on peroxisomal refolding in either control or chaperone-depleted cells. The chaperone requirement for post-heat shock refolding of peroxisomal luciferase in control cells is thus unusual in that it can be augmented by DNAJB1 or HSPB1 but not by HSPA1A; in dnHSF1 expressing cells, expression of none of the (co)-chaperones tested was effective, and an as yet to be identified, HSF1-regulated function is required.