The molecular mechanism of DNA injury by mild heat was investigated using matched isogenic mutants of E. coli. On heating at 52 degrees C for 1 h, the number of DNA single-strand breaks (SSBs) detected by the alkaline sucrose gradient sedimentation technique was consistently smaller in mutants NH5016 and BW2001, both deficient in the AP (apurinic/apyrimidinic) endonuclease of exonuclease III, as compared with their wild-type parent AB1157. The greater number of SSBs in the wild type was accompanied by more extensive cell death as compared with the AP-deficient mutants. Heating of endonuclease-free DNA systems, viz., T4 phage and T4 DNA, at 52 degrees C for up to 4 h did not result in any detectable SSB. Apparently, cellular injury by mild heat is self-inflicted through an AP-endonuclease-mediated process and hence depends on the cell's genetic complement of AP endonuclease. Mild heat is believed to activate the nucleolytic attack, and the resultant DNA-strand breaks, if not repaired, will eventually lead to cell death.