The ability of F1 (H-2k/d) responder cells to generate cytotoxic effector cells against parental and F1 targets was investigated under limiting dilution conditions. One hit limiting dilution curves, consistent with the generated cytolytic cells being the progeny of a single precursor cell, were reproducibly obtained if a culture was scored as "responding" on the basis of its ability to lyse one or more of a panel of Con A blast targets consisting of self F1 and both parents. The calculated frequency of the precursor cell in a nylon wool-nonadherent splenic responder cell population ranged from about 1/500 to 1/1600 in (B10.BR X B10.D2)F1 and (RNC X BALB/c)F1 mice. The patterns of lysis expressed by individual microcultures against the panel of F1 and both parental targets were unusual; besides patterns that were consistent with predictions of classic transplantation rules (i.e., lysis of F1 targets plus one/both parental targets), "anomalous" patterns (i.e., lysis of F1 targets or one/both parental targets exclusively) were also seen. The precursor cells that gave rise to cytolytic cells were devoid of T cell markers, whereas the cytolytic effector cells were Thy-1+, Lyt-2+, as defined by sensitivity to treatment with monoclonal antibody plus complement. The data are discussed in the context of early in vitro development of cytolytic T lymphocytes, with particular emphasis on a possible manifestation of an early T cell repertoire.