A spontaneously occurring, nalidixic acid-resistant (NalR), thermotolerant (T/r) mutant of Escherichia coli was isolated. Bacteriophage P1-mediated transduction showed that NalR mapped at or near gyr A, one of the two genes encoding DNA gyrase. Expression of gyrA+ from a plasmid rendered the mutant sensitive to nalidixic acid and to high temperature, the result expected for alleles mapping in gyrA. Plasmid linking number measurements, made with DNA from cells grown at 37 degrees C or shifted to 48 degrees C, revealed that supercoiling was about 12% less negative in the T/r mutant than in the parental strain. Each strain preferentially expressed two different proteins at 48 degrees C. The genetic and supercoiling data indicate that thermotolerance can arise from an alteration in DNA gyrase that lowers supercoiling. This eubacterial study, when coupled with those of archaebacteria, suggests that DNA relaxation is a general aspect of thermotolerance.