Limiting dilution analysis was used to estimate the frequency of human peripheral blood T lymphocytes that proliferate in response to in vitro immunization with keyhole limpet hemocyanin (KLH). Antigen-reactive cells (ARC) were estimated 9 days after primary immunization with KLH. The ARC frequency of lymphocytes from 12 subjects ranged from 1:23,800 to 1:52,631. Lymphocytes from five of these subjects were also primed for 12 days with KLH, rechallenged in secondary culture with fresh adherent cells and KLH, and assayed 4 days later. The ARC frequency increased to 1:1,123 to 1:7,247, indicating that T cell clones responsive to KLH had expanded during primary culture. In addition, we observed that the proliferative response of lymphocytes from 5 of the 12 subjects were inhibited at high cell concentrations. Depletion of OKT8+ T cells before culturing with KLH however did not alter the inhibitory effect of high concentrations of T cells.