Reovirus mRNAs are efficiently translated within host cells despite the absence of 3' polyadenylated tails. The 3' nontranslated regions (3'NTRs) of reovirus mRNAs contain sequences that exhibit a high degree of gene-segment-specific conservation. To determine whether the 3'NTRs of reovirus mRNAs serve to facilitate efficient translation of viral transcripts, we used T7 RNA polymerase to express constructs engineered with full-length S4 gene cDNA or truncation mutants lacking sequences in the 3'NTR. Full-length and truncated s4 mRNAs were translated using rabbit reticulocyte lysates, and translation product 3 was quantitated by phosphorimager analysis. In comparison to full-length s4 mRNA, translation of the s4 mRNA lacking the 3'NTR resulted in a 20 to 50% decrease in 3 produced. Addition to translation reactions of an RNA oligonucleotide corresponding to the S4 3'NTR significantly enhanced translation of full-length s4 mRNA but had no effect on s4 mRNA lacking 3'NTR sequences. Translation of s4 mRNAs with smaller deletions within the 3'NTR identified a discrete region capable of translational enhancement and a second region capable of translational repression. Differences in translational efficiency of full-length and deletion-mutant mRNAs were independent of RNA stability. Protein complexes in reticulocyte lysates that specifically interact with the S4 3'NTR were identified by RNA mobility shift assays. RNA oligonucleotides lacking either enhancer or repressor sequences did not efficiently compete the binding of these complexes to full-length 3'NTR. These results indicate that the reovirus S4 gene 3'NTR contains a translational operator sequence that serves to regulate translational efficiency of the s4 mRNA. Moreover, these findings suggest that cellular proteins interact with reovirus 3'NTR sequences to regulate translation of the nonpolyadenylated reovirus mRNAs.