Late in the infection of monkey cells by simian virus 40 (SV40), half of the viral late (L) DNA strand is transcribed into RNA sequences, referred to here as dRNA sequences, which remain in the nucleus [Khoury, G., Howley, P., Nathans, D., and Martin, M. A. (1975), J. Virol. 15, 433–437]. In the current experiments the synthesis and possible degradation of the dRNA sequences were analyzed by performing pulse-chase experiments involving [ 3H]uridine, extracting the total cellular RNA, and subsequently hybridizing this RNA against the L strands of restriction endonuclease fragments of SV40 DNA that specifically hybridize to either dRNA or mRNA sequences. Minimum estimates for the relative rate of synthesis of the dRNA sequences, compared to that of the mRNA sequences, were obtained from the relative amounts of radioactivity in these sequences after labeling cells for 10 min. These minimum estimates ranged from 0.32 to 0.55 and depended both on the individual labeling experiment and on which DNA fragments were used to assay for the dRNA and mRNA sequences. The results also showed that the dRNA sequences are eventually degraded to a size where they are no longer recoverable in hybridizable form. Sedimentation analysis in DMSO of pulse-labeled RNA did not reveal more than minor amounts of labeled multilength transcripts of the L-DNA strand in contrast to a previous analysis [Acheson, N., Buetti, E., Scherrer, K., and Weil, R. (1971), Proc. Nat. Acad. Sci. USA 68, 2231–2235] of polyoma RNA.