In Escherichia coli, the ability to elicit a heat shock response depends on the htpR gene product. Previous work has shown that the HtpR protein serves as a sigma factor (.sigma.32) for RNA polymerase that specifically recognizes heat shock promoters (A.D. Grossman, J. W. Erickson, and C.A. Gross Cell 38:383-390, 1984). In the present study we showed that .sigma.32 synthesized in vitro could stimulate the expression of heat shock genes. The in vitro-synthesized .sigma.32 was found to be associated with RNA polymerase. In vivo-synthesized .sigma.32 was also associated with RNA polymerase, and this polymerase (E.sigma.32) could be isolated free of the standard polymerase (E.sigma.70). E.sigma.32 was more active than E.sigma.70 with heat shock genes; however, non-heat-shock genes were not transcribed by E.sigma.32. The in vitro expression of the htpR gene required E.sigma.70 but did not require E.sigma.32.