The use of the phospholipid fatty acid (PLFA) composition of microorganisms to detect previous self-heating events was studied in naturally self-heated peat and in peat incubated under temperature-controlled conditions. An increased content of total PLFAs was found in self-heated peat compared to that in unheated peat. Two PLFAs, denoted T1 and T2, were detected only in the self-heated peat. Incubation of peat samples at 25 to 55 degrees C for 4 days indicated that T1 and T2 were produced from microorganisms with different optimum temperatures. This was confirmed by isolation of bacteria at 55 degrees C, which produced T2 but not T1. These bacteria produced another PLFA (denoted T3) which coeluted with 18:1omega7. T2 and T3 were identified as omega-cyclohexyltridecanoic acid and omega-cyclohexylundecanoic acid, respectively, indicating that the bacteria belonged to the genus Alicyclobacillus: T1 was tentatively identified as omega-cycloheptylundecanoic acid. T2 was detected 8 h after the peat incubation temperature was increased to 55 degrees C, and maximum levels were found within 5 days of incubation. The PLFA 18:1omega7-T3 increased in proportion to T2. T1 was detected after 96 h at 55 degrees C, and its level increased throughout the incubation period, so that it eventually became one of the dominant PLFAs after 80 days. In peat samples incubated at 55 degrees C and then at 25 degrees C, T1 and T2 disappeared slowly. After 3 months, detectable levels were still found. Incubation at 25 degrees C after heating for 3 days at 55 degrees C decreased the amounts of T2 and 18:1omega7-T3 faster than did incubation at 5 degrees C. Thus, not only the duration and temperature during the heating event but also the storage temperature following heating are important for the detection of PLFAs indicating previous self-heating.