The protease encoded by the human immunodeficiency virus (HIV) is essential for the processing of viral polyproteins encoded by the gag and pol genes into mature viral proteins. The 99-residue protease from HIV-1 contains two cysteine residues (Cys-67 and Cys-95), both of which are usually conserved in viruses isolated from patients. Despite this conservation, neither residue is required for enzymatic activity. Certain site-specific cysteine mutants of HIV-1 protease are catalytically active, and the protease from HIV-2 lacks both cysteines. Copper is a potent inhibitor of HIV-1 protease, but not of mutants lacking cysteine (A. R. Karlström and R. L. Levine, 1991, Proc. Natl. Acad. Sci. USA 88, 5552-5556). The addition of copper to the protease at pH 5.5 induced aggregation of the protein, providing a possible basis for the inhibitory action of copper. However, addition of both copper and dithiothreitol still led to inhibition of activity but did not cause aggregation. These findings led to a study of the reactivity of the cysteine residues to 5,5'-dithiobis-(2-nitrobenzoic acid) (Ellman's reagent), a sulfhydryl compound which reacts with the ionized form of cysteine residues. At pH 6.2 in 6 M guanidine, no derivatization of cysteine residues occurred, consistent with the typical pKa of cysteine expected for the denatured protein. However, in the same buffer without guanidine, the native protease reacted rapidly with concomitant loss of proteolytic activity. Peptic mapping demonstrated that both Cys-67 and Cys-95 were derivatized. A catalytically active fusion protein of protease with protein A domains was then studied with the expectation that access to Cys-95 would be hindered. This was confirmed, with only Cys-67 reacting rapidly with Ellman's reagent. Enzymatic activity was again lost, indicating that derivatization of the surface-accessible Cys-67 was sufficient to inactivate the enzyme. The reactivity and accessibility of these residues suggest an interesting approach for the development of protease inhibitors which are not directed to the substrate-binding site.