The Lyt phenotype of T cells at different stages of response to mutant H-2K antigens was determined by immunofluorescence using monoclonal rat anti-Lyt antibodies. Previous observations indicated a differential expression of the two allelic forms of Lyt-1 antigen on these cells. Since the rat antibodies recognize nonpolymorphic framework determinants of Lyt antigens, in our approach the expression of both Lyt-1 alleles was analyzed with the same antibody. It was found that cells reacting to three different H-2K mutants have the Lyt-1,2 phenotype, regardless of the Lyt-1 allele carried by the responder strain. The Lyt phenotype of responder cells remained unchanged after priming in vivo. However, cells recovered from cultures after secondary stimulation in vitro were mainly Lyt-2, with few Lyt-1,2 and virtually no Lyt-1 cells present. This change of Lyt phenotype ran in parallel with the loss of proliferative capacity to the priming antigen, but cytolytic activity of the cells remained unimpaired. Long-term proliferation of T cells induced against mutant H-2K antigens could only be maintained in the presence of a T cell growth factor. Cultures with growth factor contained almost exclusively Lyt-2 cells and exerted strong cytolytic activity. These results demonstrate that the Lyt differentiation pathway of anti-mutant T cells is from Lyt-1,2 to Lyt-2. Furthermore, the data suggest that no helper cells are induced in response to mutant H-2K antigens. A model which incorporates these findings into current concepts of T cell differentiation is discussed.