The expression of the pol gene of human immunodeficiency virus type 1 occurs via a ribosomal frameshift between the gag and pol genes. The resulting protein, a Gag-Pol polyprotein, is produced at a level 5 to 10% of that of the Gag protein. The Gag-Pol polyprotein is incorporated into virions and provides viral protease, reverse transcriptase, and integrase, which are essential for infectivity. It is generally believed that the Gag-Pol polyprotein is incorporated into virions via interaction with the Gag protein, although the details of the mechanism are unknown. To further study this problem, we have constructed a human immunodeficiency virus type 1 proviral genome which overexpresses the Gag-Pol polyprotein (Pr160gag-pol). Transfection of this proviral genome (pGPpr-) into COS-1 cells resulted in the expression of full-length Pr160gag-pol polyprotein. Although the majority of the Pr160gag-pol was confined to the cells, low levels of reverse transcriptase activity were detectable in the cell supernatants. The cotransfection of pGPpr- with a second plasmid which expresses only the Pr55gag precursor (pGAG) resulted in a significantly higher level of Pr160gag-pol in the medium of transfected cells. Sedimentation analysis using sucrose density gradients demonstrated that most Pr160gag-pol was found in fractions corresponding to the density of virion particles, indicating that the Pr160gag-pol polyprotein was released in association with a Pr55gag viruslike particle. To further characterize the requirements for the release, a mutation was constructed to express an unmyristylated Pr160gag-pol polyprotein. Coexpression with Pr55gag demonstrated that the unmyristylated Pr160gag-pol was also incorporated into virion particles. Subcellular fractionation experiments revealed that the distributions of the Pr160gag-polmyr- and Pr160gag-pol in the membrane and cytosol were similar under low- or high-ionic-strength conditions. Taken together, these results suggest that myristylation of the Pr160gag-pol polyprotein is not required for the interaction with the Pr55gag necessary for packaging into a viruslike particle.