Two global suppressors (Val331Ala & Ala334Val) have been identified for temperature sensitive folding (tsf) mutations in gene 9 of bacteriophage P22 (B. Fane, Ph.D. thesis, M.I.T.). We have introduced 19 different single amino acid substitutions at 331 and 334 independently. Folding and maturation patterns of various substitutions at two global suppressor sites indicate that 331 is located at protein surface and 334 is in the hydrophobic region. In combination with a tsf mutation, tsfH304 (Gly244Arg), only those substitutions that have similar side-chain properties to the original suppresser sequences were active as a tsf suppressor : Gly at 331 and Ile at 334. These newly identified suppressors could also alleviate the tsf defect of two other mutations. In separation of a tsf mutation, these suppressors as well as original global suppressors behaved like the wild type. No evidence for involvement of the molecular chaperonin, GroE, was found in tsf suppression of global suppressors. The suppressors may act a) by generating a site for recognition of a yet unknown chaperonin or b) by increasing the stability of an intermediate in the productive folding pathway.