Replication of human immunodeficiency virus type 1 (HIV-1) in non-dividing cells critically depends on import of the viral pre- integration complex into the nucleus. Genetic evidence suggests that viral protein R (Vpr) and matrix antigen (MA) are directly involved in the import process. An ill vitro assay that reconstitutes nuclear import of HIV-1 pre-integration complexes in digitonin-permeabilized cells was used to demonstrate that Vpr is the key regulator of the viral nuclear import process, Mutant HIV-1 pre-integration complexes that lack Vpr failed to be imported in vitro, whereas mutants that Lack a functional MA nuclear localization sequence (NLS) were only partially defective, Strikingly, the import defect of the Vpr(-) mutant was rescued when recombinant Vpr was readded, In addition, import of Vpr virus was rescued by adding the cytosol of HeLa cells, where HIV-1 replication had been shown to be Vpr- independent, In a solution binding assay, Vpr associated with karyopherin alpha, a cellular receptor for NLSs. This association increased the affinity of karyopherin alpha for basic-type NLSs, including that of MA, thus explaining the positive effect of Vpr on nuclear import of the HIV-1 preintegration complex and BSA-NLS conjugates, These results identify the biochemical mechanism of Vpr function in transport of the viral pre-integration complex to, and across, the nuclear membrane.