The cellular basis of the cytolytic cross-reactivity observed in primary allogeneic (C56BL/6 anti-DBA/2 and C57BL/6 anti-C3H/He) mixed-leukocyte cultures (MLC) was investigated by analysis of the specificity of clonal progeny derived from individual cytolytic T lymphocyte (CTL) precursor cells (CTL-P) contained within these populations. A sensitive mixed-leukocyte microculture (micro-MLC) technique was used with limiting dilution analysis by Poisson statistics to determine the frequency of CTL-P reactive against both specific and third-party (P815 and AKRA) target cells, to calculate the probability that each micro-MLC was a clone derived from a single CTL-P, and to examine the specificity of each micro-MLC assayed separately against both target cells. A total of 287 phenotypically specific, heteroclitic, and cross-reactive micro-MLC from the 2 different strain combinations were observed with a relative frequency of 81, 11, and 8%, respectively, and were calculated to have mean clone probability of 90 and 99% when based, respectively, upon the frequencies of CTL-P reactive against the specific and third-party target cells. These clones were estimated to have an approximate size of 6 X 10(4) cells, which corresponded to roughly 16 cell doublings during the 7 d of culture. 22 clones were successfully subcloned and in virtually every case, the subclones retained the specificity phenotype of the original clone from which they were derived. These results provide direct evidence for three phenotypically distinct sets of CTL as the cellular basis of cross-reactivity in MLC populations assayed against two different target cells.