The effect of intracellular pH (pHi) on heat shock protein (HSP) synthesis was investigated in C6 rat glioma cells. pHi changes were analysed by means of fluorescence spectroscopy in a perfused monitoring system allowing continuous measurements before, during and after treatments. HSP induction was determined by means of Western blots and autoradiographs. A 20 min heat shock (HS) of 44 degrees C decreased the pHi from 7.36 to 7.05 during exposure  and elicited the synthesis of heat shock proteins 2-8 h later. A pHi decrease, brought about by low extracellular pH (pHe) of 4.5 and 5.0 or 5.5, induced HSP synthesis after 1 h or 3 h, respectively. During these treatments, pHi decreased to values significantly lower than that caused by HS. Three h exposure to pHe 6.2, however, was not inductive. These results indicate that the heat shock-induced pHi decrease alone is not sufficient to stimulate HSP synthesis. In order to investigate the effect of alkaline pHi on the induction of HSP by heat, pHi was increased prior to HS treatments. Preincubation of cells at pHe ranging from 6.8 to 8.0 had little effect on pHi and on HSP synthesis. A shift of pHi to more alkaline values was achieved by adding the H+/Na+ exchanger monensin at alkaline pHe. Twenty microM monensin raised the pHi and inhibited the HSP induction depending on the pHe values: as pHe was increased from pH 7.2 to 8.0 HSP synthesis was increasingly inhibited. Monensin also diminished the HS-induced drop of pHi particularly at higher pHe. The result showed that neither a lower pHi nor a drop of pHi during HS is a necessary prerequisite for the induction, whereas alkalosis inhibits the synthesis of HSP.