We have analyzed the structure and expression of the HIV-1 vif and vpr mRNAs. The results revealed that the predominant vif and vpr mRNAs belong to the intermediate size class of HIV-1 mRNAs and that their expression is dependent on the presence of Rev protein. In addition, low levels of a small multiply spliced vpr mRNA were produced by HIV-1. cDNA cloning and expression of vpr cDNAs in eucaryotic cells revealed that high levels of Vpr were produced only from the intermediate-size mRNA in the presence of Rev. Thus, as demonstrated for the viral structural proteins, expression of Vif and Vpr is regulated by Rev. The arrangement of the splice sites and the Rev-RRE interaction are responsible for the regulation of viral expression, and especially for the switching from an early stage, producing only or primarily Tat, Rev, and Nef from multiply spliced mRNAs, to a late stage, leading to the production of Gag, Pol, Env, Vpu, Vif, and Vpr from unspliced and partially spliced mRNAs.