[3H]Uracil-pulse-labeled RNA from Escherichia coli infected with f1 bacteriophage was fractionated on polyacrylamide gels containing urea. Eight phage-specific RNA species were present with approximate lengths ranging from 2100 to 400 nucleotides. The amount of the seven largest species was increased when the infected bacteria were incubated at 41 degrees C. When the RNA was isolated and used as message in an in vitro protein-synthesizing system, most of the RNA species appeared to direct the synthesis of the phage gene VIII protein. The six largest species also directed the synthesis of the phage gene V protein. Some of the labeled smaller RNA species increased in amount after addition to rifampicin, suggesting that they may have resulted from cleavage of larger RNA species. These particular smaller RNA species also were present in infected bacteria containing a mutant RNase III. The data are discussed in terms of the regulation of synthesis of the phage-specific proteins.