Bacteriphage T7 RNAs have been fractionated on preparative polyacrylamide gels. The in vitro coding capacities of the RNAs have been determined by translation of the RNAs in a cell-free system and analysis of the polypeptide products on sodium dodecyl sulfate polyacrylamide slab gels. The T7 early RNAs are fractionated according to their molecular weight and without intermolecular aggregation. Fractionation of the late T7 RNAs gives rise to 10 major RNAs, ranging in size from 0.29 X 10(6) daltons to 2.05 X 10(6) daltons. Five of these RNAs are polycistronic and overlapping species are present for some T7 proteins. In particular, the gene 10 protein, the major capsid protein, is translated from at least three mRNAs. The smallest of these gene 10 mRNAs is monocistronic. A second gene 10 mRNA also codes for the gene 9 protein, and a third gene 10 mRNA codes for both gene 8 and gene 9 proteins. The T7 gene 3.5 protein, a T7 lytic enzyme, is also translated from several differently sized mRNAs. Comparison with published data on in vitro transcription by T7 RNA polymerase suggestes that transcription from multiple initiation sites and cleavage of larger precursors are both involved in generating the late T7 transcripts we observe. The overlapping mode of transcription could serve to amplify certain gene products.