Temperature-sensitive mutations in the coat protein of phage P22 severely restrict formation of infectious particles at restrictive temperature. A set of 25 temperature-sensitive strains, which had been localized to regions of the coat gene (Casjens, S., Eppler, K., Sampson, L., Parr, R., and Wyckoff, E. (1991) Genetics 127, 637-647), define 17 sites of single amino acid substitutions by DNA sequencing. Particles assembled from the mutant proteins at permissive temperature were not thermolabile at restrictive temperature, nor defective in the infectious process. At restrictive temperature, ts mutant polypeptide chains were synthesized at near wild-type rates. These inactive chains were not degraded within the cells. The ts chains failed to interact with scaffolding proteins to form the procapsid precursor shell, and they did not polymerize with each to form aberrant shells. Rather, the mutant coat protein accumulated as insoluble aggregates, with the amorphous morphology of inclusion bodies. The results indicate that the chains fail to reach the conformation needed for subunit-subunit or subunit-scaffolding interaction. These mutations appear to be of the class of temperature-sensitive folding mutations, which destabilize an intermediate in the intracellular folding pathway.