Viral components cross the nuclear membrane occurs at numerous steps in the life cycle of the human immunodeficiency virus (HIV) and other retroviruses. Once in the cytoplasm, HIV RNA is reverse-transcribed into double-stranded DNA which must enter the nucleus. Other retroviral regulatory proteins also enter the nucleus perform their function, and viral transcripts are exported out to the cytoplasm. The viral proteins, rev and tat, have been identified as a key regulators of the transcription and transport of HIV envelope mRNA out of the nucleus. We examined the process of nuclear protein import in several in vitro systems. I will focus on studies with the HIV rev protein. The rev protein actively shuttles between nucleolus and cytoplasm and mediates premature export of unspliced retroviral RNAs. Using digitonin permeabilized cultured cells, we developed an in vitro assay for studying both nuclear transport and nucleolar accumulation of molecules such as HIV Rev. The localization of nuclear shuttling proteins such as HIV rev is controlled by the relative rates of nuclear import and export. We also have developed an in vitro assay for nuclear export using the green fluorescent protein-labelled rev fused to a hormone- inducible import signal (Rev/Gr/GFP). Addition of steroid to cells induced nuclear import and nucleolar accumulation of the chimeric protein. Steroid removal switched off import, thus allowing direct visualization of the rev export pathway in living cells and reconstitution of the process in vitro. We examined the requirements for nuclear transport and nucleolar accumulation of HIV Rev in vitro and in living cells. This allowed a dissection of nuclear import, nucleolar accumulation and nuclear export of molecules such as HIV rev containing multiple targeting motifs. We are testing the hypothesis that Nuclear Import and Export are differentially regulated by the intracellular mediators GTP and Ca+2. We developed an in vitro assay for nuclear export of HIV rev and demonstrated that the requirements for nuclear protein export are distinct from those for nuclear import. Demonstration that Nuclear Import and Export have different requirements for Ca+2 and GTP provides a direct link between signal transduction pathways and the modulation of nuclear transport.