In vitro polyclonal activation of peripheral blood mononuclear cells (PBMCs) from 70% of the simian immunodeficiency virus (SIV) serum enzyme-linked, immunosorbent assay (ELISA)–negative sooty mangabeys leads to synthesis and release of low but significant and reproducible levels of SIV-reactive antibodies, as determined by ELISA and Western blot analysis. The predominant isotype of SIV-reactive antibodies in the pokeweed mitogen (PWM) supernatant fluids from serum ELISA-negative mangabeys is IgM, whereas the predominant isotype of SIV-reactive antibodies in seropositive mangabeys is IgG. Depletion of CD8+cells led to a marked increase in the levels of SIV-reactive antibodies detected in supernatant fluids from PWM-induced cultures from the serum ELISA-negative mangabeys. No evidence for such SIV-reactive antibodies has been found, to date, in similar unfractionated or CD8+T-cell-depleted PWM-induced PBMC cultures from uninfected macaques. Supernatant fluids from PWM cultures of PBMCs from a select group of serum ELISA-negative mangabeys, when concentrated five times, were shown to give a Western blot profile against SIV, similar to the profile seen with plasma from seropositive infected macaques and mangabeys. Evidence is presented to show that these serum ELISA-negative mangabeys are most likely latently infected with SIV. This evidence, which was obtained in samples from such ELISA-negative mangabeys, includes the detection of reverse transcriptase activity and the presence of SIV p27 in supernatant fluids of phytohemagglutinin-stimulated PBMCs in vitro. In addition, the data show the presence of CD8+T cells that regulate SIV-specific Ig synthesis and show the detection ofgagsequences by the polymerase chain reaction. Thus, the PWM assay described herein may provide a valuable additional tool for detection of lentivirus infection before or in the absence of seroconversion.