Astrocytes are cellular targets for the human immunodeficiency virus (HIV) that limit virus production, owing, at least in part, to the diminished functionality of the viral post-transcriptional stimulatory factor Rev. To understand the trafficking process in astrocytes, we compared nucleocytoplasmic transport of Rev and various proteins with well-characterized nucleocytoplasmic transport features in human astrocytes and control cells (HeLa). Localization and trafficking characteristics of several cellular and viral proteins, as well as nuclear trafficking of classical peptide signals upon microinjection were similar in both cell types, indicating maintenance of general features of nucleocytoplasmic transport in astrocytes. Quantification of fluorescence in living cells expressing Rev fused to green fluorescent protein (GFP) indicated a strong shift in intracellular distribution of Rev in astrocytes, with 50-70% of Rev in the cytoplasm, whereas the cytoplasmic proportion of Rev in HeLa cells is around 10%. The dynamics of nucleocytoplasmic trafficking of Rev were compared in astrocytes and Rev-permissive cells by monitoring migration of Rev-GFP in cell fusions using highly sensitive time-lapse imaging. Nuclear uptake of Rev was dramatically retarded in homo-polykaryons of astrocytes compared with control cells. Diminished nuclear uptake of Rev was also observed in hetero-polykaryons of Rev-permissive cells and astrocytes. These results indicate that astrocytes contain a cytoplasmic activity that interferes with nuclear uptake of Rev. Our studies suggest a model in which Rev is prevented from functioning efficiently in astrocytes by specific alterations of its nucleocytoplasmic trafficking properties.