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The heat shock response of Myxococcus xanthus was investigated and characterized. When shifted from 28 to 40°C, log-phase cells rapidly ceased growth, exhibited a 50% reduction in CFU, and initiated the synthesis of heat shock proteins (HTPs). Heat-shocked log-phase M. xanthus cells labeled with [35S]methionine were found to produce 18 major HTPs. The HTPs, analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography, were characterized with regard to molecular mass, subcellular location (periplasm, membrane, or cytoplasm), and temperature required for expression. Most HTPs were expressed at 36°C, the optimum growth temperature of M. xanthus. Cells preincubated at 36°C for 1 h before being shifted to 40°C demonstrated increased thermotolerance compared with cells shifted directly from 28 to 40°C. The HTPs produced by heat-shocked starvation-induced fruiting cells and glycerol-induced sporulating cells were also analyzed and characterized. Thirteen HTPs were detected in fruiting cells shifted from 28 to 40°C. Six of these HTPs were not seen in vegetative M. xanthus cells. Log-phase cells induced to sporulate by the addition of glycerol produced 17 HTPs after being shifted to 40°C. These HTPs were found to be a mixture of HTPs detected in heat-shocked log-phase cells and heat-shocked fruiting cells.