The aim of this study was to investigate the presence and the fine specificity of anti-CD4 autoantibodies in seronegative subjects sexually exposed to HIV-1. Anti-CD4 autoantibodies were previously detected in a fraction of HIV-1-seropositive individuals. Whole sera, purified IgG fractions, and supernatants of EBV-transformed lymphoblastoid cell lines were analyzed by means of ELISA, Western blot, and by competition assays using monoclonal antibodies with known fine specificities. Anti-CD4 antibodies were found in 6 of 18 individuals exposed to HIV-1 infection and who have been persistently seronegative. These antibodies inhibited HIV-1-driven syncytium formation, did not interfere with the CD4-gp120 interaction, and competed for CD4 binding with two of three anti-CD4 monoclonals with known fine specificities. Moreover, autoantibodies with the same fine specificities were found in the supernatants of oligoclonal EBV-transformed B cell lines derived from these individuals. At variance, in the HIV-1-positive patients included in our study, the anti-CD4 antibody response was directed to a broader panel of epitopes, including those involved in CD4-gp120 interactions. In conclusion, anti-CD4 antibodies specific for defined epitopes of the CD4 molecule are generated in the course of an early immune response to HIV-1 antigens in the absence of other signs of infection, as they can be detected by conventional methods. These autoantibodies may play a protective role either alone or in association with other cellular and humoral factors.