The temperature-sensitive (ts) Chinese hamster ovary (CHO) cell mutant tsHl contains a therrnolabile leucyl-tRNA synthetase. Upon incubation at the nonpermissive temperature of 39.5"C, the enzyme became reversibly inhibited over a period of minutes, and the cells lost viability over a period of many hours. However, killing of tsH1 by acute heating at 45°C was identical to that of wild-type (SC) cells. In addition, the heat-induced inhibition of protein synthesis was similar for both cell types, as measured after acute heating at 45°C. Furthermore, both killing and inhibition of protein synthesis showed thermotolerance in both cell types. In contrast to the effects at 4 5 T , at 39.5"C, neither the inhibition of leucyl-tRNA synthetase activity nor the killing of tsHl expressed thermotolerance. Also, treatment of tsHl at 39.5"C did not induce thermotolerance to killing at 45°C. The inhibition of leucyl-tRNA synthetase activity in tsH1 at 39.5"C was further distinguished from the 45°C-induced inhibition of protein synthesis in SC cells by a much more rapid reversal of the inhibition of leucyl-tRNA synthetase activity. Also, the rate of reversal of the inhibition of protein synthesis by 45°C in SC cells was decreased by increased heat dose. Such was not true for the 39.5"C inhibition of leucyl-tRNA synthetase activity in tsH1. The data indicate that there exist two distinct types of thermal inhibition-one slowly reversible type which was observed during and after heating at 45°C and both induced and expressed thermotolerance, and a second, rapidly reversible type, which was evident only during heating of tsHl at 39.5"C and neither induced nor expressed thermotolerance.