The synthesis of phiX174-specific RNA has been studied in infected cells in which the thymine of the viral (+) strand of the parental RF(*), of the complementary (-) strand of the parental RF, or of both strands of the progeny RF molecules has been replaced with 5-bromouracil (5 BU). By irradiation of such cells with UV light at a wavelength of 313 nm it was possible to affect, specifically, the 5 BU-labeled strands. When the progeny RF molecules contain thymine, irradiation has no effect upon the synthesis of viral-specific RNA, regardless of 5 BU substitution in either strand of parental RF. If, however, progeny RF is labeled with 5 BU, irradiation produces a major decrease of viral RNA synthesis. It is concluded that many progeny RF molecules can serve as templates for transcription at late times of infection. Irradiation, prior to RF replication, of cells in which, particularly, the complementary strand of RF contains 5 BU, appears to decrease the ability of the parental RF to replicate.