We have previously documented the appearance of auto-anti-idiotypic (anti-Id) antibody in the serum of normal subjects after booster immunization with tetanus toxoid (TT) antigen. In the present study circulating auto-anti-Id-bearing cells and the regulatory role of auto-anti-Id antibody on anti-TT antibody synthesis were examined. Two adult volunteers were boosted with TT antigen, and IgG (Fab')2 anti-TT was prepared from plasma obtained 7 and 10 days after boosting. Auto-anti-Id antibody directed against autologous IgG (Fab')2 anti-TT became detectable in the serum starting 2 wk and up to 4 mo after booster immunization with TT. Circulating cells capable of specifically binding fluorescein-conjugated autologous IgG (Fab')2 anti-TT were identified starting 3 wk after immunization and throughout the 3- to 4-mo study period. Auto-anti-Id-bearing cells were predominantly B cells and secreted auto-anti-Id antibody after stimulation with pokeweed mitogen (PWM) in vitro. When added to cultures of peripheral blood lymphocytes (PBL) obtained 10 days after immunization, serum auto-anti-Id antibody inhibited the PWM-induced synthesis of IgG anti-TT. This inhibition was idiotype specific and affected predominantly the synthesis of anti-TT idiotypes recognized by the auto-anti-Id antibody. Auto-anti-Id antibody did not affect the synthesis of IgG anti-TT by PBL that co-circulated with the auto-anti-Id antibody. These results suggest that auto-anti-Id antibodies play a role in the regulation of the normal human immune response.