The HIV-1 replication cycle requires the nucleocytoplasmic export of intron-containing viral RNAs, a process that is ordinarily restricted. HIV overcomes this by means of the viral Rev protein, which binds to an RNA secondary structure called the Rev response element (RRE) present in all unspliced or incompletely spliced viral RNA transcripts. The resulting mRNP complex is exported through interaction with cellular factors. The Rev-RRE binding interaction is increasingly understood to display remarkable structural plasticity, but little is known about how Rev-RRE sequence differences affect functional activity. To study this issue, we utilized a lentiviral vector assay in which vector titer is dependent on the activity of selected Rev-RRE pairs. We found that Rev-RRE functional activity varies significantly (up to 24-fold) between naturally occurring viral isolates. The activity differences of the Rev-RRE cognate pairs track closely with Rev, but not with RRE activity. This variation in Rev activity is not correlated with differences in Rev steady state protein levels. These data suggest that Rev sequence differences drive substantial variation in Rev-RRE functional activity between patients. Such variation may play a role in viral adaptation to different immune milieus within and between patients and may be significant in the establishment of latency. The identification of differences in Rev-RRE functional activity in naturally occurring isolates may also permit more efficient production of lentiviral vectors.