Temperature sensitive folding (tsf) mutations in gene 9 of bacteriophage P22 interfere with the folding and association of the tailspike polypeptide chain at restrictive temperature. Two global suppressors (Val331Ala & Ala334Val) have been identified by genetic analysis (B. Fane, Ph.D. thesis, M.I.T.). Characterizing the suppression mechanism of tsf is crucial for understanding how polypeptides fold in vivo. We have introduced 16 distinct single amino acid substitutions at 331 by cassette mutagenesis. Effect of residue substitutions at 331 on folding and activity of tailspike was examined. Most substitutions except Pro and Lys were allowed at 331 position. 331Pro substitution behaved like a tight is and 331Lys was lethal at all temperature. When combined with tsH304 (Gly224 → Arg), most of substitutions at 331 except Ala and Gly could not restore wild type property completely. Unexpectedly Pro at 331 (by itself is ts) could suppress stH304. The result suggests that in the course of in vivo polypeptide folding, some readjustment occurs at secondary structure interaction.