We have analyzed the accumulation of viral genomic and messenger RNAs in tissue culture cells during the first 24 hr of acute infection with lymphocytic choriomeningitis virus (LCMV). This has allowed comparison of the relative amounts of the genomic L and S RNAs (both genomic sense and genomic complementary sense) and of nucleoprotein (NP) and glycoprotein precursor (GP-C) mRNAs. Using these techniques NP mRNA was detected simultaneously with genomic S RNA, but the amount of NP mRNA accumulating during this period of infection was higher than that of GP-C mRNA. This is consistent with a model for ambisense RNA transcription and replication proposed by D. D. Auperin, V. Romanowski, M. Galinski and D. H. L. Bishop (J. Virol. 52:897-904,1984). The accumulation of S RNA exceeded that of L RNA and, for both L and S RNAs, the amount of genomic sense RNA was higher than that of genomic complementary RNA.