The prevalence of antibodies against HIV-1 regulatory proteins in sera of HIV-infected patients from different stages of disease was investigated. HIV-1 vif, tat, and nef genes were cloned in procaryotic vectors and were expressed as MS-2 fusion proteins (vif and nef) or as a non-fusion protein (tat). These recombinant proteins were employed in immunoblot experiments. The specificity of the recognition was confirmed by competition experiments and with control sera from HIV-negative patients. Analysis of 136 serum samples revealed a high percentage of antibodies against nef, irrespective of the stage of disease. Antibodies against tat were found less frequently and increased from 16% to 40% with disease progression. Vif antibodies were detected only in a low percentage in early stages of disease, but their prevalence increased to 36% and 72% with progression of disease to AIDS-related complex and AIDS. Our data suggest that the detection of antibodies against nef may represent an additional and useful marker for the diagnosis of HIV infection, whereas the detection of vif antibodies may indicate disease progression.