Mice immunized with hapten-autologous serum albumin conjugates (DNP-mouse serum albumin) were shown to contain immune B and T cells with specificity for the conjugate. Fractionation on antigen-coated Sepharose beads showed that B cells could be subdivided in two major groups: those reacting against the haptenic group (DNP) and those reactive against the new antigenic determinants (NADs) introduced into the protein carrier by the hapten coupling. It was shown previously that humoral antibodies formed against hapten-mouse serum albumin conjugates also were directed against these two groups of antigenic determinants and that the immune response to the NADs does not follow the genetic rules of high or low response against the hapten used. All together these findings support the distinct nature of the NADs over the haptenic groups, as recognized both at the humoral and cellular level. Absorption of mouse cells immune to hapten-autologous serum albumin conjugates on antigen-coated Sepharose beads using a variety of incubation conditions resulted in no specific retention of T cells. Therefore we had to resort to specificity studies of T cells in relation to T cell function. Relatively pure immune T cell suspensions were obtained using fractionation on anti-immunoglobulin-coated columns. DNP-MSA-specific T cells were shown to be very specific for the DNP-MSA conjugate with only one exception: they cross-reacted with antigenic determinants on DNP-rat serum albumin. As DNP-specific help was excluded in the present transfer system (as shown by the inability of cells from DNP-skin-painted mice and DNP-heterologous protein conjugate specific T cells [anti-immunoglobulin column purified] to help a DNP-MSA response), these results demonstrate the NAD specificity of the DNP-MSA-reactive T cells. The cross-reactivity pattern of DNP-MSA-specific T cells was similar to that found for humoral anti-NAD antibodies produced against the same immunogen. Whether B and T cells are activated by the same antigenic determinants is discussed.