A major limitation in the simian immunodeficiency virus (SIV) system has been the lack of reagents with which to identify the antigenic determinants that are responsible for eliciting neutralizing antibody responses in macaques infected with attenuated SIV. Most of our information on SIV neutralization determinants has come from studies with murine monoclonal antibodies (MAbs) produced in response to purified or recombinant SIV envelope proteins or intact SIV-infected cells for relatively short periods of time. While these studies provide some basic information on the potential immunogenic determinants of SIV envelope proteins, it is unclear whether these murine MAbs identify epitopes relevant to antibody responses elicited in monkeys during infection with either wild-type or attenuated SIV strains. To accomplish maximum biological relevance, we developed a reliable method for the production of rhesus monoclonal antibodies. In the present study, we report on the production and characterization of a unique panel of monoclonal antibodies derived from four individual monkeys inoculated with SIV/17E-CL as an attenuated virus strain at a time when protective immunity from pathogenic challenge was evident. Results from these studies identified at least nine binding domains on the surface envelope glycoprotein; these included linear determinants in the V1, V2, cysteine loop (analogous to the V3 loop in human immunodeficiency virus type 1), and C5 regions, as well as conformational epitopes represented by antibodies that bind the C-terminal half of gp120 and those sensitive to defined mutations in the V4 region. More importantly, three groups of antibodies that recognize closely related, conformational epitopes exhibited potent neutralizing activity against the vaccine strain. Identification of the epitopes recognized by these neutralizing antibodies will provide insight into the antigenic determinants responsible for eliciting neutralizing antibodies in vivo that can be used in the design of effective vaccine strategies.