We examined the effects of reducing agents on the expression of heat shock protein 27 (hsp27), alpha B crystallin, and hsp70 in C6 rat glioma cells in response to stress. Cells were exposed to arsenite (100 microM for 1 h) in the presence of dithiothreitol at various concentrations (0.03-2 mM), and the accumulation of all three proteins was markedly stimulated in cells that had been exposed to arsenite in the presence of a low concentration (0.03-0.1 mM) of dithiothreitol. Stimulation of these arsenite-induced responses was also observed in the presence of 0.1 mM 2-mercaptoethanol or 0.05 mM dithioerythritol. The enhanced expression of mRNAs for hsp27, alpha B crystallin and hsp70, as well as the prolonged activation of heat shock transcription factor 1 (HSF1), were also observed in cells that had been treated with arsenite in the presence of 0.05 mM dithiothreitol. The arsenite-inducible expression of the three proteins was completely suppressed when dithiothreitol was present at concentrations above 1 mM during the stress period, although delayed activation of the binding to a heat shock element (HSE) by phosphorylated HSF was observed in these cells. Exposure of cells first to arsenite for 1 h and then to dithiothreitol resulted in a very effective suppression of the arsenite-inducible responses, and the responses were inhibited even by a low concentration of dithiothreitol. These results suggest that the signal transduction pathway for the arsenite-induced expression of hsps involves at least two redox-sensitive steps: (i) a process that is stimulated by mild reducing power during the stress period; and (ii) a process that is followed by the activation of HSF and is very sensitive to suppression by a reducing agent.