Viral components must be transported across 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 then enters the nucleus. A growing amount of evidence points to the viral matrix protein of HIV as a mediator of the nuclear transport event. The matrix protein has been isolated in recombinant form and its nuclear localization examined. Other retroviral regulatory proteins also enter the nucleus to 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. These proteins contain sequences that are necessary and sufficient for targeting to the nucleolus. An in vitro system using permeabilized cells has been developed to examine the nucleolar targeting of proteins bearing such targeting signals. We have observed that transport to the nucleolus is an active process. In addition to its nucleolar transport rev is actively exported from the nucleus. While a specific signal mediating nuclear export of rev has been identified the mechanism of protein export is poorly understood. To examine this nuclear export event we have constructed a chimeric protein consisting of rev coupled to the green fluorescent protein (GFP). The chimera also contains the steroid binding domain of the glucocorticoid receptor thus allowing precise control over nuclear import. Addition of hormone leads to nuclear transport; removal of hormone initiates export but does not allow subsequent import. These approaches should allow us to analyze and biochemically dissect the nuclear export process.