The Nef protein plays a major role in vivo in promoting HIV and SIV replication and pathogenesis. In vitro, Nef has been shown to down-regulate cell surface molecules, such as CD4 and MHC-I, alter T cell signaling, and enhance virion infectivity. These effects are attributed to interactions of Nef with cellular proteins. In addition, HIV Nef is incorporated into viral particles, mainly localizing in the virion cores. However, no report has been published to date regarding Nef interactions with virion proteins. By immunoprecipitation, Nef was found to bind to viral enzymes. Using yeast two-hybrid and GST pulldown procedures to find out direct potential partners of Nef, Nef was consistently found to interact with viral integrase (IN). The interaction between Nef and IN was stronger when Nef was present as the viral protease-cleaved isoform. We hypothesize that the interaction of Nef with viral integrase or other virion proteins may explain the presence of Nef in viral cores. In addition, this interaction suggests that Nef may accompany the reverse transcription and the preintegration complexes during the early steps of the infection cycle and potentially affect infectivity during these steps.