Escherichia coli strain CR341T28 will not grow at temperatures above 34 degrees C in liquid medium, and the adenylate kinase of this strain is heat sensitive. When a culture was shifted from a permissive (30 degrees C) to a nonpermissive (36 degrees C) temperature, the adenylate energy charge fell from 0.9 to 0.2, with a concurrent decrease in the number of viable cells and in the specific activity of adenylate kinase. When cultures of the temperature-sensitive strain were grown at temperatures above 30 degrees C, the adenylate energy charge, the specific activity of adenylate kinase, and the growth rate were lower than the corresponding parameters for the parental strain. By isotopic labeling of the adenine nucleotides in vivo, it was determined that increasing growth temperatures between 30 and 34 degrees C for the heat-sensitive strain resulted in a decrease in the adenosine triphosphate-to-adenosine monophosphate and adenosine triphosphate-to-adenosine diphosphate ratios. Between 26 and 30 degrees C the adenosine triphosphate-to-adenosine diphosphate ratio was essentially normal in the temperature-sensitive strain, but the adenosine triphosphate-to-adenosine diphosphate ratio was decreased. The adenylate ratios in the parental strain did not change between 30 and 34 degrees C. The adenylate kinase mass action ratio for each strain was essentially constant under all growth conditions. When assayed at 30 degrees C, the affinities of the enzyme from the mutant strain were somewhat lower than those of the parent adenylate kinase. The mutant enzyme also did not exhibit the substrate inhibition that was observed at high adenosine monophosphate concentrations with the parental enzyme. An increase in the assay temperature from 30 degrees to 40 degrees C had little or no effect on the Km values determined for the parental adenylate kinase, but caused the Km values determined for the mutant adenylate kinase to increase by a factor of two or more.