Gene 14 is a recently discovered late gene of phage P22, mapping between the DNA injection and head completion genes (P. Youderian and M. Susskind (1980), Virology 107, 258-269). The gene 14 product has not been detected in phage particles. We have studied the defective phenotype of amber mutants in gene 14 to determine the role of gp14. The yield of physical particles from 14- infections is normal, but the infectivity of those particles is reduced by 60-80%. The noninfectious particles adsorb to but do not kill the host cell, as if they were defective in DNA injection. No differences in morphology, DNA composition, DNA permutation, or protein composition have been detected between 14- and wild-type particles. Procapsids, the capsid precursor to DNA packaging, exhibit a similar reduction in viability when isolated from 14- infected cells, assayed by in vitro DNA packaging. This is consistent with the gene 14 product functioning in the assembly or maturation of the procapsid. The three DNA injection proteins, encoded by genes 7, 16, and 20, are assembled into the particle at the procapsid stage. The defect in 14- particles may arise from improper organization or modification of one or more of the three proteins needed for DNA injection.