(1) Treatment of resident peritoneal macrophages for 8 h with macrophage colony-stimulating factor (M-CSF) increased release of superoxide anion (O2-) stimulated by phorbol 12-myristate 13-acetate. Gel electrophoresis of pulse-labelled proteins with L-[35S]methionine showed that a number of proteins were induced during activation by M-CSF. Immunoblot analysis with antibody against heat shock protein (HSP) 90, HSP70, or HSP60 demonstrated that M-CSF induced these stress-inducible HSPs; the timing of induction and level of each HSP correlated with the increase in O2- production. The activated macrophages acquired resistance to H2O2-induced damage. M-CSF also stimulated the synthesis of a heat shock cognate protein (HSC70); however, the induction occurred at 1 h, when O2- production was not yet augmented, but at which time L-[35S]methionine incorporation into cell proteins was already enhanced. (2) Gel mobility shift assay with oligonucleotide coding for the heat shock element showed that M-CSF activated the heat shock factor within 15 min, and the activation continued for at least 8 h. Northern-blot analysis with a cDNA probe for human HSP70 or HSC70 showed that accumulations of HSP70 and HSC70 mRNAs coincided with the inductions of the respective proteins. (3) These results suggest that M-CSF may induce the transcriptional activation of heat shock genes, and that the stress-inducible HSPs as well as HSC70 may play an important role in the activation of macrophages by functioning as molecular chaperones and by protecting the macrophage against the auto-oxidative damage associated with the respiratory burst.