The process of RNA incorporation into nascent virions is thought to be critical for efficient retroviral particle assembly and production. Here we show that human immunodeficiency virus type 1 mutant particles (which are highly unstable and break down soon after release from the cell) lacking nucleocapsid (NC) core protein-mediated RNA incorporation are produced efficiently and can be recovered at the normal density when viral protease function is abolished. These results demonstrate that RNA binding by Gag is not necessary for retroviral particle assembly. Rather, the RNA interaction with NC is critical for retroviral particle structural stability subsequent to release from the membrane and protease-mediated Gag cleavage. Thus, the NC-RNA interaction, and not simply the presence of RNA, provides the virus with a structural function that is critical for stable retroviral particle architecture.