The antibody response to structural and regulatory viral proteins was studied in 14 rhesus (Macaca mulatta) and 6 cynomolgus (Macaca fascicularis) macaques experimentally infected with HIV-2 or SIVMAC. To investigate the humoral antibody response to the negative regulatory factor (nef), the recombinant protein was expressed to high levels with recombinant vaccinia virus (VV). nef-specific antibodies were detected in 14 of 20 infected macaques (70%). In sera of all infected monkeys antibodies directed to the structural proteins gp120, p56, and p24 appeared 2 to 6 weeks postinfection. In contrast, the extent and the appearance of nef-specific antibodies during the course of infection varied considerably between individual animals. However, only in sera of four animals (20%) were nef-specific antibodies detectable as early as those against the core proteins p24 and p56. In SIVMAC-infected rhesus macaques at different clinical stages, the antibody response towards nef neither correlated with the development of viral latency nor to disease progression or viremia. Our data indicate that in macaques experimentally infected with SIV or HIV-2 antibody formation against nef is not a useful diagnostic marker either for early detection of viral infection or of disease progression.