Objective The purpose of this study was to assay reactivity of antibody-positive sera to different parts of the HIV-1 and HIV-2 proteinase (PR) proteins. Design Since the majority of HIV-1-antibody-positive sera react to the proteinase, but the antigenic determinants on the protein have not been identified, we attempted to identify these determinants. Interventions We synthesized 18 peptides representing the PR of HIV-1 and HIV-2 in order to map serum reactivity to the PR protein. Results Both HIV-1-and HIV-2-antibody-positive sera recognized four distinct antigenic regions in the HIV-1 and HIV-2 PR. Conclusions Correlation between our results and the crystallographic structure of the protein revealed that the antigenic regions are positioned at the surface of the HIV-1 PR. Although the structure of HIV-2 PR has not yet been characterized, our results indicate that the folding of the HIV-1 and HIV-2 PR may be very similar.