A synthetic rev gene containing substitutions which introduced unique restriction sites but did not alter the deduced amino acid sequence was used as a vehicle to construct mutations in rev. Insertion or substitution mutations within a domain of Rev resulted in proteins able to inhibit the function of Rev protein in trans. Rev function was monitored in a cell line, HLfB, which contained a rev- mutant provirus. HLfB cells require the presence of rev for virus production, which was conveniently monitored by immunoblot detection of p24gag. Trans-dominant mutants were identified after expression in bacteria and delivery into HLfB cells by protoplast fusion. In addition, the trans-dominant phenotype was verified by expression of the mutant proteins in HLfB cells after cotransfection. These studies define a region between amino acid residues 81 and 88 of rev, in which different mutations result in proteins capable of inhibiting Rev function.