The effects of slow chilling (2 degrees C min(-1)) and rapid chilling (2,000 degrees C min(-1)) were investigated on the survival and membrane fluidity of Escherichia coli, of Bacillus subtilis, and of Saccharomyces cerevisiae. Cell death was found to be dependent on the physiological state of cell cultures and on the rate of temperature downshift. Slow temperature decrease allowed cell stabilization, whereas the rapid chilling induced an immediate loss of viability of up to more than 90 and 70% for the exponentially growing cells of E. coli and B. subtilis, respectively. To relate the results of viability with changes in membrane physical state, membrane anisotropy variation was monitored during thermal stress using the fluorescence probe 1,6-diphenyl-1,3,5-hexatriene. No variation in the membrane fluidity of all the three microorganisms was found after the slow chilling. It is interesting to note that fluorescence measurements showed an irreversible rigidification of the membrane of exponentially growing cells of E. coli and B. subtilis after the instantaneous cold shock, which was not observed with S. cerevisiae. This irreversible effect of the rapid cold shock on the membrane correlated well with high rates of cell inactivation. Thus, membrane alteration seems to be the principal cause of the cold shock injury.