A system for excess DNA hybridization of Moloney murine leukemia virus (M-MuLV)-specific RNA from infected mouse cells with M-MuLV cDNA immobilized on nitrocellulose filters was developed. In the presence of unlabeled heterologous rabbit liver RNA, 0.3-0.5% of labeled, infected cell nuclear RNA bound to the filters, while 0.05% or less of nuclear RNA from uninfected cells bound. Sedimentation analysis of pulse-labeled nuclear RNA was performed, and hybridization across sucrose gradients indicated that the major pulse-labeled, virus-specific RNA was 38S, similar or identical in sedimentation to the virion subunit RNA. A minor component of pulse-labeled, virus-specific RNA larger than 38S, was detected (40-60S), but kinetic experiments indicated that it was not an obligate precursor to 38S virus-specific RNA. Simultaneous analysis of steady state and pulse-labeled, virus-specific nuclear RNA across sucrose gradients indicated that the 38S virus-specific RNA was not detectably different from the steady state "35S" nuclear RNA previously identified. More detailed resolution on agarose gels also showed no difference. Thus the primary transcript of M-MuLV-specific RNA appears to be 38S, the same size as stable cellular virus-specific RNA, and no evidence for a higher molecular weight precursor was found.