T cells from guinea pigs immunized with the hapten 2,4-dinitrophenyl (DNP)-coupled directly to mycobacteria are of interest since they recognize and respond to DNP conjugated to many but not all carriers. The experiments reported here further analyze the structure of the complex, chemically defined antigenic determinants recognized by such T cells. These antigenic determinants can have DNP coupled either to the xi-amino group of lysyl residues or to the hydroxyl group of tyrosyl residues. Furthermore, essential contributions to the determinant recognized by such T cells are made by amino acid residues to which the hapten is not attached. Such residues are thought to be close to the hapten group itself, since introducing a small spacer between hapten and carrier prevents recognition. The hapten itself is also recognized and discriminated from other haptens with great precision by these T lymphocytes. The strain of guinea pig immunized affects the precise specificity characteristics of the responding T cells, in a way that may reflect the activity of histocompatibility-linked immune response genes. Finally, the characteristics of the immunogen have been studied. It is thought that the lipid content of the mycobacteria may be critical in inducing the hapten-reactive T cells, and this is supported by finding similar responses in T cells from guinea pigs immunized with DNP protein to which lipid has been covalently attached. Thus, the T-cell population being studied, while recognizing haptens with great precision, appears to require a larger determinant for activation than do hapten-specific B lymphocytes.