The steady-state distribution of translating ribosomes on the reovirus serotype 1 Lang strain polycistronic s1 mRNA and the monocistronic s4 mRNA was mapped using a sensitive primer extension assay and cDNA clones of the S1 and S4 genes. The distribution of translating ribosomes on the reovirus s1 and s4 mRNAs was not uniform. Major positions of ribosome pausing were detected in both rabbit reticulocyte and wheat germ cell-free protein synthesizing systems programmed with wild-type and site-directed mutant mRNAs. Two of the ribosome pause sites represent initiation and termination, rate-limiting steps of translation. For the polycistronic s1 mRNA, analysis of mutants in which either the sigma 1 ORF1 initiation codon or the sigma 1NS ORF2 initiation codon was eliminated by site-directed mutagenesis unequivocally established the identity of the specific ribosome pauses with specific ORF translational events. Ribosomes were far less uniformly distributed along the overlapping ORF regions of the polycistronic s1 mRNA than they were along the ORF of the monocistronic s4 mRNA. Furthermore, the rate-limiting initiation event at the sigma 1NS ORF2 AUG led to ribosome stacking and elongation arrest in the sigma 1 ORF1. These results begin to provide a conceptual framework for the dynamics of translation of complex as compared to simple viral mRNAs and suggest that ribosome activity may vary at multiple discrete regions on an mRNA.