Recent investigations in mice revealed that natural immunological tolerance to endogenous minor lymphocyte-stimulating locus 1a (MIs-1a) antigen correlates primarily with deletion of MIs-1a-specific V beta 6+ T lymphocytes in the thymus. Similar mechanisms account for acquired tolerance in some instances since the neonatal injection of MIs-1a-expressing MHC compatible cells in neonatal mice within the first 24 h of life causes clonal deletion of V beta 6+ T cells. Here we demonstrate that V beta 6+ T cells are not deleted in mice neonatally treated with MIs-1a spleen cells expressing allogeneic H-2 molecules. However, when such non-deleted V beta 6+ T cells were tested in vitro, no interleukin 2 (IL-2) secretion or proliferation was observed after MIs-1a stimulation. This non-responsive state could be overcome by addition of exogenous IL-2, consistent with the fact that V beta 6+ cells enlarged and expressed IL-2 receptors upon MIs-1a stimulation. Furthermore, the same neonatally treated mice showed in vitro functional unresponsiveness of cytotoxic T cells but not of IL-2-secreting cells specific for the tolerated allogeneic MHC antigens. Taken together, our data indicate that neonatal tolerance to MIs-1a can be accomplished by either clonal deletion or clonal anergy, and that it does not necessarily correlate with tolerance to MHC determinants.