We have employed site-directed mutagenesis to evaluate the contribution of the amino acid residues, Glu³⁴, Arg³⁷, and Lys³⁸, to the thermostability of the thermophilic protein, Bacillus stearothermophilus DNA binding protein HU (BstHU), relative to the mesophilic homologue, Bacillus subtilis HU (BsuHU). Mutants BstHU-E34D and BstHU-K38N, in which Glu³⁴ Lys³⁸ were individually replaced by the corresponding residues, Asp³⁴ and Asn³⁸, in BsuHU, exhibited decreased thermostabilities by 2. 08 and 2. 17 kJ/mol, respectively, whereas mutant BstHU-R37K with Lys instead of Arg at position 37 showed no change in thermostability. These results suggested that Glu³⁴ and Lys³⁸ contribute to the thermostability of BstHU by forming a possible salt bridge on the hydrophilic surface. The role of Glu³⁴ as a salt bridge partner was corroborated by generating BstHU mutant protein BstHU-E34Q, in which the Glu residue was changed to Gln; this substitution clearly decreased the stability of the protein by 2. 71 kJ/mol. The contribution of this favorable salt bridge to the thermostability was further investigated as the salt and pH dependencies of the stabilities of the wild-type BstHU, BstHU-K38N, and BstHU-E34D. As for salt dependency, the stability of the wild-type relative to those of the two mutants decreased with an increase in ionic strength, indicating that the electrostatic interaction between Glu³⁴ and Lys³⁸ indeed significantly contributes to the thermostability of BstHU. As for pH dependency, the difference in thermostability between the wild-type BstHU and mutant BstHU-K38N showed that the mutant protein was as stable as the wild-type protein at pH 2. 0, however, at neutral pH, the mutant protein was less stable than the wild-type protein. In contrast, the difference between the melting temperatures of mutant BstHU-E34D and the wild-type did not change as a function of pH. This suggested that the Glu³⁴ residue may play an important role in the thermostability not only as a partner in the salt bridge with Lys³⁸, but also by stabilizing the α-helix from residue 18 to 35 in BstHU. On the basis of the present results, together with the previous results [Kawamura et al. (1996) Biochemistry 35, 1195-1200], three mutations, Glu¹⁵ to Gly, Asp³⁴ to Glu, and Asn³⁸ to Lys, were simultaneously introduced into mesophilic protein BsuHU to demonstrate their contributions to thermostability. This combination of multiple thermostabilizing mutations generated a more stable mutant protein HU, showing a T_m value of 64. 5°C compared to 48. 6°C for the wild-type BsuHU.