The Pseudomonas phaseolicola bacteriophage phi6 incorporated labeled UTP into an acid-insoluble precipitate. Incorporation was dependent on the presence of manganese acetate, ATP, GTP, CTP, and a short heat treatment of the phage; the reaction was stimulated by NH(4)Cl. The substitution of (14)C-ATP, -CTP or -GTP for UTP, together with the appropriate unlabeled ribonucleoside triphosphates, disclosed that CMP was incorporated to the greatest extent followed by GMP, UMP, and AMP. Radioactive RNAs formed by the reaction were resistant to RNases A and T(1) in high salt but susceptible to these nucleases in low salt. The labeled RNA co-sedimented and co-electrophoresed with phi6 double-stranded (ds) RNA. However, the distribution of the radioactivity into the three ds-RNA components varied depending on the (14)C-ribonucleoside triphosphate used in the reaction. The incorporation of UMP was primarily into the two smaller ds-RNA segments, GMP primarily into the large ds-RNA segment, and CMP and AMP were about equally distributed into all three ds-RNA segments.