Four phage phi29 suppressor-sensitive mutants of cistron O have been examined for production of 14C-labeled viral-specific proteins in restrictive infections of Bacillus subtilis and fail to produce the protein of the viral neck lower collar. Cistrons O and F have been placed on the genetic map, containing 12 cistrons, by three-factor crosses. The phenotypes of five cistron J mutants have been analyzed by sodium dodecyl sulfate gel electrophoresis and autoradiography, and in three instances fragments of the normal polypeptide were detected. Three factor crosses with these mutants and a virus with a clear plaque phenotype were used to initiate the mapping of cistron J and the determination of the orientation of transcription in this map region.