A frameshift event is necessary for expression of the products of the pol gene in a number of retroviruses, including human immunodeficiency virus type 1 (HIV-1). The basic signals necessary for frameshifting consist of a shifty sequence in which the ribosome slips and a downstream stimulatory structure which can be either a stem-loop or a pseudoknot. In HIV-1, much attention has been paid to the frameshift site itself, and only recently has the role of the downstream structure been examined. Here we used a luciferase-based experimental system to analyze in vivo the cis and trans factors potentially involved in controlling frameshifting efficiency at the gag-pol junction of HIV-1. We demonstrated that high-level frameshifting is dependent on the presence of a palindromic region located downstream of the site where the frameshift event takes place. Frameshifting efficiencies were found to be identical in mouse fibroblasts and the natural host cells of the virus, i.e., CD4+ human lymphoid cells. Furthermore, no increase in frameshifting was observed upon virus infection. Previous observations have shown that viral infection leads to specific alteration of tRNAs involved in translation of shifty sites (D. Hatfield, Y.-X. Feng, B.J. Lee, A. Rein, J.G. Levin, and S. Oroszlan, Virology 173:736-742, 1989). The results presented here strongly suggest that these modifications do not affect frameshifting efficiency.