Heat-shock proteins (Hsp's) are a family of molecular chaperones that contribute to protection from environmental stress. In this report, we demonstrate that a member of this family, Hsp70, facilitates nuclear import of HIV-1 preintegration complexes (PICs). The mechanism of this activity appears to be similar to the one used by Vpr, an HIV-1 protein regulating viral nuclear import and replication in macrophages. Indeed Hsp70 stimulated binding of HIV-1 matrix antigen to GST–karyopherin α fusion protein and rescued nuclear import of a Vpr-defective HIV-1 strain in vitro. Binding studies with truncated forms of GST–karyopherin α demonstrated that both Vpr and Hsp70 bind to a region in the amino-terminal part of the karyopherin α molecule. This region appears to be distinct from the binding sites for two other karyopherin α cargoes, basic-type NLS-containing proteins and transcription factor STAT-1. Vpr competed with Hsp70 for binding to karyopherin α. These results suggest the presence of a novel regulatory site on karyopherin α which is used by Hsp70 and Vpr to stimulate interaction between the HIV-1 PIC and karyopherin α and thus promote viral nuclear import.