All retroviruses contain two copies of genomic RNA that are linked noncovalently. The dimeric RNA of human immunodeficiency virus type 1 (HIV-1) undergoes rearrangement during virion maturation, whereby the dimeric RNA genome assumes a more stable conformation. Previously, we have shown that the packaging of the HIV-1 polymerase (Pol) proteins reverse transcriptase (RT) and integrase (IN) is essential for the generation of the mature RNA dimer conformation. Analysis of HIV-1 mutants that are defective in processing of Pol showed that these mutant virions contained altered dimeric RNA conformation, indicating that the mature RNA dimer conformation in HIV-1 requires the correct proteolytic processing of Pol. The HIV-1 Pol proteins are multimeric in their mature enzymatically active forms; RT forms a heterodimer, and IN appears to form a homotetramer. Using RT and IN multimerization defective mutants, we have found that dimeric RNA from these mutant virions has the same stability and conformation as wild-type RNA dimers, showing that the mature enzymatically active RT and IN proteins are dispensable for the generation of mature RNA dimer conformation. This also indicated that formation of the mature RNA dimer structure occurs prior to RT or IN maturation. We have also investigated the requirement of Pol for RNA dimerization in both Mason-Pfizer monkey virus (M-PMV) and Moloney murine leukemia virus (MoMuLV) and found that in contrast to HIV-1, Pol is dispensable for RNA dimer maturation in M-PMV and MoMuLV, demonstrating that the requirement of Pol in retroviral RNA dimer maturation is not conserved among all retroviruses.