Two different limiting dilution systems have been applied to compare precursor frequencies of alloreactive cytotoxic T cells (CTL-P) in the polyclonally and specifically activated lymphocyte populations and in selected Lyt T cell subsets. Both systems make use of T cell growth factor for T cell expansion but differ with respect to the activation step in that lymphocytes are either activated directly with allogenetic stimulator cells or are sensitized polyclonally with concanavalin A (Con A) in bulk culture before their expansion under limiting dilution conditions. In polyclonally activated C57BL/6 lymphocyte populations, two types of CTL-P specific for H-2d alloantigens could be identified: a frequent set with a frequency of 1/100-1/300, and a rare set with a frequency of 1/2,000-1/8,000. In contrast, only a single CTL-P set was found in specifically activated populations with a frequency similar to that of the frequent CTL-P found on Con A blasts. In Con A blasts, the frequent at higher cell concentrations by suppressor T cells, whereas rare CTL-P were insensitive to this suppressive mechanism. Whereas in specifically activated T cells, the predominant CTL-P phenotype was Lyt-123, the predominant Lyt phenotypes for the frequent and the rare CTL-P found in Con A blasts were Lyt-123 and Lyt-123, respectively, which suggests that they represent primary and secondary CTL-P, respectively. The results are discussed with respect to previous reports on the involvement of Lyt T cell subsets in the generation of cytotoxic responses and their regulation by T suppressor cells.