The effects of MOI and of virion age on the RNA species from Moloney murine leukemia virus (MLV) and Moloney murine sarcoma-leukemia virus (MSV[MLV]) were investigated by electrophoresis on 2.4% polyacrylamide gels in the presence of 0.2% sodium dodecyl sulfate (SDS). The native RNA from MLV consisted of a species with an apparent molecular weight of 6.5 × 106 as well as low-molecular-weight material, and the profiles obtained were independent of MOI and virion age. Heat-denatured RNA from 5-min MLV consisted primarily of a species with an apparent molecular weight of 3.4 × 106 whereas that of 16-hour MLV consisted of a heterogeneous, continuous distribution of sizes ranging from 3.4 × 106 to 1.0 × 105 daltons. The nature of the RNA profiles for MSV (MLV) was dependent on the infection conditions used to produce the virus. If low MOI conditions were used, the RNA patterns were identical to those observed for the RNA of MLV alone, and the ratio of MLV/MSV in such stocks was approximately 60 as determined by bioassays. If high MOI conditions were used, the native RNA consisted of two high-molecular-weight classes, one having an apparent molecular weight of 6.5 × 106 and the other an apparent molecular weight of 4.0 × 106. Upon incubation of the native RNA at 80° for 3 min, both classes were converted to species with a molecular weight of 1.8 × 106. High multiplicity passage conditions resulted in virus stocks with much smaller MLV/MSV ratios (approximately 2) and also with much lower titers than stocks produced using low MOI.