Cell-free translation in the presence of pancreatic microsomal membranes of the full-length envelope transcript of the human immunodeficiency virus type 1 (HIV-1) yielded the expected extensively glycosylated and immunologically reactive gp160 envelope-protein precursor. In addition to this gp160, a shorter glycoprotein, which we designated gp120*, was produced due to a premature translation arrest. Utilizing kinetic experiments, pulse-chase analyses and various gp160 envelope RNA mutants, we demonstrated that the in-vitro-produced gp120* was not formed by cleavage of the gp160 precursor or by internal initiation of translation. A gp120 produced before gp160 synthesis was completed, and, independent of the gp160 proteolytic processing, has been shown to be produced and sequestered in the endoplasmic reticulum of HIV-1-infected cells [Willey, R. L., Klimkait, T., Frucht, D. M., Bonifacino, J. S. & Martin, M. A. (1991) Virology 184, 319-329]. The specific translational arrest shown to occur in vitro was found to be dependent on the Rev-responsive element, since deletion of this highly structured sequence abolished the production of gp120*. We found that the combination of two contiguous putative stem loops of the Rev-responsive element, located at nucleotides 7494-7522 and 7525-7550 of the HIV-1 Rev-responsive-element sequence, was responsible for the production of this truncated protein. To our knowledge, these stem-loop structures, distinct from that known to bind the Rev protein, represent the first example responsible for the production of alternative products by premature translational arrest in higher eukaryotes.