The regulatory protein Vpr of the human immunodeficiency virus HIV-1 performs multiple functions during the HIV replicative cycle. It is involved in the transport of the viral preintegration complex into the nucleus, and has the ability to interact with nuclear proteins such as transcription factors and cyclin-dependent kinases. In this study we examine for the first time the kinetics of intranuclear binding and accumulation at the nuclear envelope of fluorescently labelled full-length Vpr in vitro. We show that intranuclear binding is strongly dependent on the presence of cytosolic factors; in the absence of cytosol, Vpr associates predominantly with the nuclear envelope. Specific regulation of the interactions of Vpr with cytosolic factors, as well as with sites at the nuclear envelope and within the nucleus, is thus implicated, but conventional nuclear transport factors such as importin alpha/beta do not appear to be involved.