The frequency of all precursors of T cells capable of proliferation (PTL-p) and of all cytotoxic T-cell clones (CTL-p) was determined for different thymocyte subpopulations using a high cloning-efficiency, Con A and growth factor driven, limit-dilution assay and a lectin-mediated, non-specific cytotoxic readout. As shown previously, more than 99% of precursors were confined to the medullary-type fraction, isolated by fluorescence activated cell sorting as the 14-15% of thymocytes showing low binding of peanut agglutinin (PNA). However, 20-50% of medullary-type cells appeared incapable of responding in a culture system allowing all peripheral T cells to grow, suggesting that the absolute size of the functional pool was 7-12% of all thymocytes. The 3-4% cortisone-resistant fraction of thymus gave a high precursor frequency (PTL-p 1 in 1.3; CTL-p 1 in 6) and a high cloning efficiency per Thy 1 positive cell (80%) which was nevertheless below that of peripheral T cells. However, only 20-25% of the total thymic PTL-p and CTL-p could be recovered in this fraction. Functional precursors were therefore within both the cortisone-sensitive and the cortisone-resistant subgroups of medullary-type thymocytes. Attempts to induce function in PNA+ cortical-type thymocytes by increasing the level of T-cell growth factors in the cultures gave only a marginal increase, the bulk of small cortical cells remained functionally inert. However, the low frequency of precursors found in the PNA+ fraction (around 1% of that in PNA- thymocytes) was not entirely due to PNA- contaminants since a PNA+, high H-2 blast fraction, representing about 4% of all thymocytes, showed a significant, although still low, PTL-p and CTL-p frequency amounting to less than 1% of the total thymus precursor pool. The relevance of this minor subset is discussed.