Mice rendered tolerant at birth of H-2 alloantigens display concordant in vivo and in vitro phenotypes: they fail to reject skin grafts bearing the tolerated antigens, and their lymphoid cells fail to participate in tolerogenspecific mixed lymphocyte reactions (MLRs) and cell-mediated lympholysis (CML). Tolerant animals normally reject third-party skin allografts and develop positive MLRs and CML to third-party antigens. It has been suggested that clonal deletion of antigen reactive cells is the basis for this spectrum of responses. To investigate further the basis for the lack of in vitro alloreactivity, we conducted limiting dilution studies with lymph node cells from adult mice tolerant of various H-2 disparities. When the frequencies of (a) cells responding to the tolerogen in MLR and (b) interleukin-2-producing cells against the tolerogen were determined, it appeared that both types of cells were functionally deleted, that is, the frequency of cells responding to tolerogen-bearing stimulator cells was identical with that of cells stimulated with syngeneic cells. On the assumption that cells from H-2 tolerant mice are deficient in helper cell activity toward the tolerogen, we performed CML cultures under conditions in which exogenous help was provided in the form of supernatants derived from concanavalin A stimulated rat spleen cell cultures. Lymphoid cells from H-2 tolerant mice generated significant cytotoxicity toward the tolerogen under these conditions, although the absolute level of killing was reduced compared with that of cells from normal mice. Limiting dilution assays confirmed that Tc precursors were present in tolerant mice, and that they were reduced to less than 10% of normal numbers; however, tolerogen-specific Tc precursors were present in frequencies significantly greater than self-reactive Tc precursors. These data indicate that a deletion mechanism operates in neonatal transplantation tolerance to reduce the clone size of all three categories of functional T cells assayed, but that its efficiency is greatest among cells destined to provide specific T-cell help. The absolute functional deletion of helper cells determines the in vitro CML unresponsiveness of lymphoid cells from tolerant mice, and may be a crucial factor in promoting the in vivo phenotype of skin allograft tolerance.