Temperature sensitive mutations of the tailspike protein of Salmonella phage P22 affect the folding and chain association at restrictive temperature. Mutant polypeptides which cannot fold and mature properly at 39℃ aggregate inside the cells. Physiological defect of these mutants is likely to be due to the destabilization of an folding intermediate. In order to examine in detail the effect of amino acid substitutions at the mutation sites, twelve different single amino acid substitutions were made at the site of a mutation, TsfU2 (Asp^(238)>Ser). Most of the substitutions except Pro and Arg did not affect folding and maturation of the tailspike protein at 28℃. However, at 39℃ only the wild type residue, Asp, and the Thr substitution allowed the formation of the tailspike trimers. The results suggest that a stereo-specific interaction of the residue 238 is required for the folding and maturation of the tailspike, which is critical for stabilizing the folding intermediate at high temperature.