Evolution depends on the acquisition of genomic mutations that increase cellular fitness. Here, we evolved Escherichia coli MG1655 cells to grow at extreme temperatures. We obtained a maximum growth temperature of 48.5 degrees C, which was not increased further upon continuous cultivation at this temperature for >600 generations. Despite a permanently induced heat shock response in thermoresistant cells, only exquisitely high GroEL/GroES levels are essential for growth at 48.5 degrees C. They depend on the presence of lysyl-tRNA-synthetase, LysU, because deletion of lysU rendered thermoresistant cells thermosensitive. Our data suggest that GroEL/GroES are especially required for the folding of mutated proteins generated during evolution. GroEL/GroES therefore appear as mediators of evolution of extremely heat-resistant E. coli cells.