The ribonucleic acid (RNA) specified by bacteriophage phi29 was isolated under conditions which minimized physical and enzymatic degradation, reduced aggregation, and enriched for completed molecules. This RNA was fractionated both by sedimentation through sucrose density gradients and electrophoresis through polyacrylamide gels to measure the size and relative amount of each component. Early RNA consisted of six components of molecular weight 0.75 x 10(6), 0.44 x 10(6), 0.37 x 10(6), 0.25 x 10(6), 0.09 x 10(6), and 0.04 x 10(6), accounting for 35% of the coding capacity of phi29 deoxyribonucleic acid (DNA). All of these components except the one at 0.44 x 10(6) were detected when infection occurred in the presence of chloramphenicol. Synthesis of the major early component (0.75 x 10(6)) ceased shortly after the onset of viral DNA synthesis. The other species of early RNA were synthesized throughout the latent period. Three additional components, 1.75 x 10(6), 0.93 x 10(6), and 0.07 x 10(6), appear at late times. The two large RNAs may be polycistronic messenger RNAs corresponding to the seven viral capsid proteins.