Synthesis of Gag-Pol polyproteins of retroviruses requires ribosomes to shift translational reading frame once or twice in a -1 direction to read through the stop codon in the gag reading frame. It is generally believed that a slippery sequence and a downstream RNA structure are required for the programmed -1 ribosomal frameshifting. However, the mechanism regulating the Gag-Pol frameshifting remains poorly understood. In this report, we have defined specific mRNA elements required for sufficient ribosomal frameshifting in equine anemia infectious virus (EIAV) by using full-length provirus replication and Gag/Gag-Pol expression systems. The results of these studies revealed that frameshifting efficiency and viral replication were dependent on a characteristic slippery sequence, a five-base-paired GC stretch, and a pseudoknot structure. Heterologous slippery sequences from human immunodeficiency virus type 1 and visna virus were able to substitute for the EIAV slippery sequence in supporting EIAV replication. Disruption of the GC-paired stretch abolished the frameshifting required for viral replication, and disruption of the pseudoknot reduced the frameshifting efficiency by 60%. Our data indicated that maintenance of the essential RNA signals (slippery sequences and structural elements) in this region of the genomic mRNA was critical for sufficient ribosomal frameshifting and EIAV replication, while concomitant alterations in the amino acids translated from the same region of the mRNA could be tolerated during replication. The data further indicated that proviral mutations that reduced frameshifting efficiency by as much as 50% continued to sustain viral replication and that greater reductions in frameshifting efficiency lead to replication defects. These studies define for the first time the RNA sequence and structural determinants of Gag-Pol frameshifting necessary for EIAV replication, reveal novel aspects relative to frameshifting elements described for other retroviruses, and provide new genetic determinants that can be evaluated as potential antiviral targets.