The expression of the pol gene of human immunodeficiency virus type 1 (HIV-1) occurs by a ribosomal frameshift between the gag and the pol genes. The Gag-Pol polyprotein is produced at levels of 5 to 10% of that of the Gag protein, and is incorporated into virions to provide the viral protease, reverse transcriptase, and integrase which are essential for replication. The mechanism(s) by which the Gag-Pol polyprotein are targeted to the HIV virion is unknown, although it is believed to be via an interaction with the Gag protein. To further explore the mechanism by which the Gag-Pol polyprotein is incorporated into virions, we have constructed a mutation which changes an aspartic acid in the protease active site to asparagine (pHXB2pro-); a four-amino-acid insertion into the protease gene (pHXB2Smal); and insertion of translational termination codons in the protease gene following the gag gene (pHXB55). Transfection of these proviral genomes into COS-1 cells resulted in intracellular expression of only Pr55gag, demonstrating the inactivation of the viral protease. The expression of Pr55gag was evident in cells transfected with pHXB2pro- during a short pulse and first 3 hr of chase period, whereas at later times the intracellular levels of Pr55gag were greatly reduced. In contrast, the intracellular Pr55gag expressed from transfection of pHXB2Smal or pHXB55 were evident even after 6- or 12-hr chase times. To ascertain the effects of the mutations on the assembly and release of viruslike particles, the supernatants from the transfected cells were analyzed for the presence of Pr55gag. The release of Pr55gag from cells transfected with pHXB2pro- occurred as early as 1 hr following chase period, and increased for up to 3 hr. In contrast, reduced levels of Pr55gag were detected in the medium from cells transfected with pHXB2Smal or pHXB55. Subcellular fractionation studies demonstrated that the Pr55gag expressed from transfection of pHXB2pro- was rapidly targeted to intracellular membranes, while the majority of the Pr55gag expressed from transfection of pHXB2Smal or pHXB55 was distributed evenly between the cytoplasm and membrane fractions. Finally, the released viruslike particles obtained from the transfection of proviral genome pHXB2pro- were stable to mild detergent treatment, whereas particles obtained from transfection of pHXB2Smal and pHXB55 were relatively unstable. These results demonstrate that subtle changes in the Gag-Pol polyprotein of HIV-1 can have significant effects on the assembly and physical stability of the released virus.