Defective variants of human immunodeficiency virus type 1 (HIV-1) protease (HIV PR) have been engineered to inhibit wild-type (wt) HIV PR activity. These variants were designed to promote the formation of heterodimers and to destabilize the formation of inactive variant homodimers of HIV-1 protease through substitutions at Asp-25, Ile-49, and Gly-50 (Babé, L. M., Rosé, J., and Craik, C. S. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 10069-10073; McPhee, F., Good, A. C., Kuntz, I. D., and Craik, C. S. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 11477-11481). The mechanism of action of these dominant-negative inhibitors was established using recombinantly expressed defective monomers. The defective monomers were refolded in vitro in the presence of wt HIV PR and showed dose-dependent inhibition of proteolytic activity. This inhibition was shown to result from the formation of inactive heterodimers between defective and wt HIV PR monomers. Heterodimer formation was detected by (i) isolating refolded, inactive heterodimers using histidine-tagged defective monomers and (ii) isolating heterodimers from bacteria coexpressing both wt and defective variants of HIV PR. Single-chain variants of HIV PR, in which the C terminus of the wt HIV PR monomer was covalently tethered to the N terminus of the defective monomer, were also expressed and analyzed. Thermal denaturation of these single-chain heterodimers using differential scanning calorimetry revealed a 1.5-7.2 degrees C greater thermal stability than single-chain wt HIV PR. The thermodynamic trend shown by these three variants mirrors their relative inhibition in provirus transfection assays. These data support the model that the effects seen both in tissue culture and in vitro arise from an increase in stability conferred on these heterodimers by interface mutations and identifies heterodimer formation as their mechanism of inhibition.