ABSTRACT Since, like other osmolytes, proline can act as a protein stabilizer, we investigated the thermoprotectant properties of proline in vitro and in vivo. In vivo, elevated proline pools in Escherichia coli (obtained by altering the feedback inhibition by proline of γ-glutamylkinase, the first enzyme of the proline biosynthesis pathway) restore the viability of a dnaK -deficient mutant at 42°C, suggesting that proline can act as a thermoprotectant for E. coli cells. Furthermore, analysis of aggregated proteins in the dnaK -deficient strain at 42°C by two-dimensional gel electrophoresis shows that high proline pools reduce the protein aggregation defect of the dnaK -deficient strain. In vitro, like other “chemical chaperones,” and like the DnaK chaperone, proline protects citrate synthase against thermodenaturation and stimulates citrate synthase renaturation after urea denaturation. These results show that a protein aggregation defect can be compensated for by a single mutation in an amino acid biosynthetic pathway and that an ubiquitously producible chemical chaperone can compensate for a defect in one of the major chaperones involved in protein folding and aggregation.