A translational frameshift is necessary in the synthesis of Escherichia coli release factor 2 (RF-2) to bypass an in-frame termination codon within the coding sequence. The nucleotide sequence preceding the in-phase stop codon within RF-2 mRNA is complementary to the 3' anti-(Shine-Dalgarno sequence) region found in prokaryotic 16S rRNA and Weiss et al. (1988) have concluded that this pairing triggers the frameshift event. In vitro production of RNA coding for RF-2, suitable for translation on eukaryotic ribosomes, has enabled testing of whether eukaryotic ribosomes can frameshift at this sequence. The 18S rRNA of eukaryotic ribosomes does not contain the 3' anti-(Shine-Dalgarno sequence) region. The prokaryotic RF-2 gene and the gene for the other release factor, RF-1, which does not contain an in-frame stop codon, were subcloned into transcription vectors such that the RNA transcripts produced in vitro would resemble a typical eukaryotic mRNA. These RF-1 and RF-2 RNAs both synthesized a major product of Mr approximately 45,000 when translated in vitro within reticulocyte lysate; the size expected for full length RF-1 and RF-2 molecules. The RF-2 product was immunoprecipitated by RF-2-specific antibodies, including those to regions of the protein encoded in the mRNA downstream from the frameshift site. The putative premature termination product, an oligopeptide of 25 amino acids, was not detected, but a chemically synthesized derivative was shown to be very unstable within the translation system. Although it was not possible therefore to calculate an absolute efficiency of frameshifting, the relative efficiency of the translation of RF-2 RNA was estimated to be 10-20% of that of RF-1 RNA in the reticulocyte system. This was similar to the relative synthesis of the two proteins in a plasmid-DNA-directed prokaryotic transcription/translation system. These results show that in vitro on eukaryotic ribosomes where the Shine-Dalgarno-type interaction is not possible, high efficiency frameshifting around the in-phase stop codon in the RF-2 mRNA can still occur.