Highly conserved among primate lentiviruses, the human immunodeficiency virus type 1 (HIV-1) Nef protein enhances viral infectivity by an unknown mechanism. Nef-defective virions are blocked at a stage of the HIV-1 life cycle between entry and reverse transcription, possibly virus uncoating. Nef is present in purified HIV-1 particles; however, it has not been determined whether Nef is specifically recruited into HIV-1 particles or whether virion-associated Nef plays a functional role in HIV-1 replication. To address the specificity and potential functionality of virion-associated Nef, we determined the subviral localization of Nef. HIV-1 cores were isolated by detergent treatment of concentrated virions followed by equilibrium density gradient sedimentation. Relative to HIV-1 virions, HIV-1 cores contained equivalent amounts of reverse transcriptase and integrase, decreased amounts of the viral matrix protein, and trace quantities of the viral transmembrane glycoprotein gp41. Examination of the particles by electron microscopy revealed cone-shaped structures characteristic of lentiviral cores. Similar quantities of proteolytically processed Nef protein were detected in gradient fractions of HIV-1 cores and intact virions. In addition, detergent-resistant subviral complexes isolated from immature HIV-1 particles contained similar quantities of Nef as untreated virions. These results demonstrate that Nef stably associates with the HIV-1 core and suggest that virion-associated Nef plays a functional role in accelerating HIV-1 replication.