A fixation and immunofluorescence staining procedure for measurements of heat shock proteins (hsp) by flow cytometry is reported. Three fixatives were compared: 80% methanol at -20 degrees C for 1 h, 70% ethanol at 0 degree C for 1 h, and 3% paraformaldehyde at 4 degrees C for 1 h followed by 0.2% NP-40. Cells fixed with methanol showed strongest immunofluorescence and lowest nonspecific fluorescence. The level of hsp 70 as a function of time after heating followed the same kinetics as the development of thermotolerance reported by others. The level of hsp 70 increased with increasing heat dose up to a maximum heat dose, and above this heat dose a decrease in the level of hsp 70 was observed. Correlated measurements of the level of hsp 70 and DNA showed that hsp 70 was found in all phases of the cell cycle. The level of hsp 70 increased about two-fold in unheated cells throughout the cell cycle. The increase in G2 + M cells compared with G1 cells was lower in cells heated at 45 degrees C for 20 min followed by incubation at 37 degrees C for 24 h before fixation and staining, than in unheated cells. The results show that flow cytometry provides a rapid and quantitative technique for measuring hsp. Correlated measurements of hsp and other cellular parameters might also be obtained.