Heat shock proteins (HSPs) are induced by various physical, chemical, and biological stresses. HSPs are known to function as molecular chaperones, and they not only regulate various processes of protein biogenesis but also function as lifeguards against proteotoxic stresses. Because it is very useful to discover nontoxic chaperone-inducing compounds, we searched for them in herbal medicines. Some herbal medicines had positive effects on the induction of HSPs (Hsp70, Hsp40, and Hsp27) in cultured mammalian cells. We next examined 2 major constituents of these herbal medicines, glycyrrhizin and paeoniflorin, with previously defined chemical structures. Glycyrrhizin had an enhancing effect on the HSP induction by heat shock but could not induce HSPs by itself. In contrast, paeoniflorin had not only an enhancing effect but also an inducing effect by itself on HSP expression. Thus, paeoniflorin might be termed a chaperone inducer and glycyrrhizin a chaperone coinducer. Treatment of cells with paeoniflorin but not glycyrrhizin resulted in enhanced phosphorylation and acquisition of the deoxyribonucleic acid-binding ability of heat shock transcription factor 1 (HSF1), as well as the formation of characteristic HSF1 granules in the nucleus, suggesting that the induction of HSPs by paeoniflorin is mediated by the activation of HSF1. Also, thermotolerance was induced by treatment with paeoniflorin but not glycyrrhizin. Paeoniflorin had no toxic effect at concentrations as high as 80 microg/ mL (166.4 microM). To our knowledge, this is the first report on the induction of HSPs by herbal medicines.