Human immunodeficiency virus (HIV) Gag precursor protein is cleaved by viral protease (PR) within GagPol precursor protein to produce the mature matrix (MA), capsid, nucleocapsid, and p6 domains. This processing is termed maturation and required for HIV infectivity. In order to understand the intracellular sites and mechanisms of HIV maturation, HIV molecular clones in which Gag and GagPol were tagged with FLAG and hemagglutinin epitope sequences at the C-termini, respectively were made. When coexpressed, both Gag and GagPol were incorporated into virus particles. Temporal analysis by confocal microscopy showed that Gag and GagPol were relocated from the cytoplasm to the plasma membrane. Mature cleaved MA was observed only at sites on the plasma membrane where both Gag and GagPol had accumulated, indicating that Gag processing occurs during Gag/GagPol assembly at the plasma membrane, but not during membrane trafficking. Fluorescence resonance energy transfer imaging suggested that these were the primary sites of GagPol dimerization. In contrast, with overexpression of GagPol alone an absence of particle release was observed, and this was associated with diffuse distribution of mature cleaved MA throughout the cytoplasm. Alteration of the Gag-to-GagPol ratio similarly impaired virus particle release with aberrant distributions of mature MA in the cytoplasm. However, when PR was inactive, it seemed that the Gag-to-GagPol ratio was not critical for virus particle release but virus particles encasing unusually large numbers of GagPol molecules were produced, these particles displaying aberrant virion morphology. Taken together, it was concluded that the Gag-to-GagPol ratio has significant impacts on either intracellular distributions of mature cleaved MA or the morphology of virus particles produced.