DafA is encoded by the dnaK operon of Thermus thermophilus and mediates the formation of a highly stable complex between the chaperone DnaK and its co-chaperone DnaJ under normal growth conditions. DafATth contains 87 amino acid residues and is the only member of the DnaKTth chaperone system for which no corresponding protein has yet been identified in other organisms and whose particular function has remained elusive. Here, we show directly that the DnaKTth-DnaJTth-DafATth complex cannot represent the active chaperone species since DafATth inhibits renaturation of firefly luciferase by suppressing substrate association. Since DafATth must be released before the substrate proteins can bind we hypothesized that free DafATth might have regulatory functions connected to the heat shock response. Here, we present evidence that supports this hypothesis. We identified the 70S ribosome as binding target of free DafATth. Our results show that the association of DafATth and 70S ribosomes does not require the participation of DnaKTth or DnaJTth. On the contrary, the assembly of DnaKTth-DnaJTth-DafATth and ribosome-DafA Tth complexes seems to be competitive. These findings strongly suggest the involvement of DafATth in regulatory processes occurring at a translational level, which could represent a new mechanism of heat shock response as an adaptation to elevated temperature.