We examined the structural stabilities after heat treatment of 22 mutants of rat prolactin (rPRL) with amino acid replacements at 15 different positions and recombinant wild-type rPRL (WT-PRL) as part of our series of studies on site-directed mutagenesis of rPRL. When WT-PRL at low concentrations (0.1 ∼ 10 ng/ ml) was heated at 100°C for 20 min, it lost its Nb2 proliferation activity, whereas at high concentrations (above 1 fig/ml), its activity remained. Temperature-dependent loss of the proliferation activity of 10 ng/ml WT-PRL after heat treatment for 5 min was observed. Next, we examined the proliferation activities of the 22 mutants heated at 60 and 70°C for 5 min. After treatment at 60°C, all the mutants retained their initial proliferation activities, whereas treatment at 70°C reduced their activities to about 63%, except for one in which glutamic acid at position 118 was replaced by lysine (E118K), suggesting that the mutations did not induce structural instability. The mutant E118K retained 84% of its initial activity after treatment at 70°C, significantly (P < 0.01) higher than the WT-PRL value. The temperature-dependency profile of the Nb2 proliferation activity of E118K also showed it had significantly increased thermo-stability. Meanwhile another mutant (E118Q) at the same residue showed no increased thermo-stability, suggesting that changing a negative charge (E) to a positive one (K) at position 118 induces ionic bond formation with a neighboring negative charge, resulting in thermostabilization of the structure of PRL.