The question of functional differentiation within the CD8 subset has been addressed in a model of TcR-transgenic (TcR-tg) mice expressing a TcR specific for H-2Kb (Ti). CD8+ Ti+ T cells present in the periphery of these mice have no cytotoxic T lymphocyte (CTL) activity unless they are stimulated with H-2Kb-expressing cells. In contrast to T cells from normal H-2k littermates, alloantigen induction of CTL from TcR-tg mice is independent of CD4+ T helper (Th) cells and is accompanied by high level secretion of interleukin-(IL)-2 by Ti+ CD8+ T cells. Precursor frequency analysis performed on CD8+ cells from TcR-tg mice revealed a high frequency of Th as compared to CTL precursors. This raised the possibility of the existence of distinct subpopulations within CD8+ precursors with different requirements for differentiation to functional CTL. FACS analyses (performed on resting and on in vitro stimulated T cells from normal and TcR-tg mice) demonstrated a heterogeneous expression of Ly-6C on CD8+ cells with a large enrichment of Ly-6C- cells among the Ti+ cells which persisted after stimulation with H-2b cells in conditions that led to a homogeneous expression of the activation markers pgp-1 and CD69. The possibility that Ly-6C expression could mark functionally different subpopulations in CD8+ T cells was investigated. Stimulation of sorted populations of Ly-6C- and Ly-6C+ cells allowed detection of CTL precursors in both these subsets and the majority of limiting dilution wells containing one pCTL also scored positive for IL-2 secretion. Thus, for CD8+ T cells expressing the same TcR, differentiation led to acquisition of both IL-2 secretion and CTL function and there was no evidence for the existence of a distinct population of helper-dependent CTL precursors.