CBA (H-2k) mice were rendered tolerant to H-2d antigens by injection of (CBA X BALB/c)F1 spleen cells at birth. At intervals of 2 days to 12 weeks, the frequencies of anti-H-2d cytotoxic T lymphocyte precursor cells (CTL-P) in thymus and spleen were determined by using a limiting-dilution microculture assay system for CTL-P. This assay, utilizing irradiated H-2d stimulator cells and concanavalin A-induced spleen cell conditioned medium, was shown to be linear over the range 30 to 100,000 responder cells and uninfluenced by IJ-positive cells. A profound and long-lasting deficit in activatable CTL-P, first demonstrable by day 5 of life in the thymus and day 8-10 in the spleen, developed in mice rendered tolerant, reaching a greater than 95% reduction by 6 weeks. Functional clonal deletion thus seems to be at least as important in the tolerant state as suppressor T cells. Repeated in vivo administration of anti-IJk serum partially inhibited clonal deletion, suggesting either that suppressor T cells are actively involved in producing clonal deletion or that IJk-bearing cells in the donor inoculum or the host represent an important factor.