We have explored the mechanism directing the intracellular trafficking and nucleolar accumulation of the human immunodeficiency virus type 1 (HIV-1) Rev protein. Treatment of Rev-expressing cells with mycophenolic acid, an inhibitor of inosine monophosphate dehydrogenase, resulted in a redistribution of Rev from the nucleoli to the nucleoplasm and cytoplasm. In contrast, a Rev effector domain mutant was retained in the nucleus, indicating the involvement of this domain in the protein's nuclear retention/nucleocytoplasmic transport. Identical results were obtained by inhibiting transcription using actinomycin D or 5,6-dichlorobenzimidazole riboside. All three drugs were found to inhibit biosynthetic labeling of ribosomal RNA and to disrupt nucleolar morphology, suggesting a correlation between nucleolar/nuclear retention of Rev, continued ribosomal RNA synthesis, and intact nucleolar architecture. Results of binding/immunofluorescence assays using isolated, permeabilized nuclei and extracts of cells expressing Rev demonstrated that the protein is able to bind to nucleoli in vitro, in the absence of active cellular processes or eukaryotic posttranslational modifications. Rev derived from actinomycin D-treated cells showed equivalent binding, indicating that the inhibitor did not directly interfere with the ability of the protein to interact with nucleolar structures. Rev's interaction with nucleoli was directed by the protein's arginine-rich RNA-binding/nucleolar localization domain, and was abrogated by pretreatment of the nuclei with RNaseA, indicating a requirement for RNA, probably ribosomal RNA.