The interaction of the human immunodeficiency virus type 1 (HIV-1) nucleoprotein complex with the cell nuclear import machinery is necessary for viral replication in macrophages and for the establishment of infection in quiescent T lymphocytes. The karyophilic properties of two viral proteins, matrix (MA) and Vpr, are keys to this process. Here, we show that an early step of HIV-1 nuclear import is the recognition of the MA nuclear localization signal (NLS) by Rch1, a member of the karyopherin-alpha family. Furthermore, we demonstrate that an N-terminally truncated form of Rch1 which binds MA but fails to localize to the nucleus efficiently blocks MA- but not Vpr-mediated HIV-1 nuclear import. Correspondingly, NLS peptide inhibits the nuclear migration of MA but not that of Vpr and prevents the infection of terminally differentiated macrophages by vpr-defective virus but not wild-type virus. These results are consistent with a model in which Rch1 or another member of the karyopherin-alpha family, through the recognition of the MA NLS, participates in docking the HIV-1 nucleoprotein complex at the nuclear pore. In addition, our data suggest that Vpr governs HIV-1 nuclear import through a distinct pathway.