Ag85B (also known as α antigen or MPT59) is immunogenic, and induces expansion and differentiation of TCRVβ11+CD4+ T cells to IFN‐γ‐producing cells in C57BL/6 (I‐Ab) mice. We reported that Peptide‐25 (amino acids 240–254) of Ag85B is a major T cell epitope, and its amino acid residues at position 244, 247, 249 and 252 are I‐Ab contact residues. Here we examined roles of IFN‐γ in the generation of Peptide‐25‐reactive CD4+ TCRVβ11+ T cells and the efficacy of mutant peptides of Peptide‐25 for Th1 development in mice other than C57BL/6 mice. Immunization of C57BL/6 mice with Peptide‐25 included in incomplete Freund’s adjuvant led to preferential induction of CD4+ TCRVβ11+ IFN‐γ‐ and tumor necrosis factor‐α‐producing T cells. Compared with other I‐Ab‐binding peptides such as Peptide‐9 of Ag85B, 50V of pigeon cytochrome c and ovalbumin (OVA)265–280 peptide, only Peptide‐25 was capable of inducing enormous expansion of TCRVβ11+ IFN‐γ‐producing T cells. Treatment of C57BL/6 mice with anti‐Vβ11 antibody before Peptide‐25 immunization reduced the development of CD4+ IFN‐γ‐producing T cells. Furthermore, B10.A(3R) mice, I‐Ab‐positive and TCRVβ11– strain, showed remarkably lower response to Peptide‐25 immunization than C57BL/6 mice. Peptide‐25‐primed IFN‐γ–/– cells showed significantly decreased expansion of CD4+ TCRVβ11+ T cells as compared with wild‐type cells. Interestingly, Peptide‐25‐primed cells from MyD88‐deficient mice responded to Peptide‐25 and differentiated into IFN‐γ‐producing cells to a similar extent as wild‐type mice, indicating Toll‐like receptor‐independent IFN‐γ production. These results imply that IFN‐γ plays important roles for the generation and expansion of CD4+ TCRVβ11+ T cells in response to Peptide‐25. Although Peptide‐25 was non‐immunogenic in C3H/HeN mice, a substituted mutant of Peptide‐25, 244D247V, capable of binding to I‐Ak, induced Th1 development. These results clearly demonstrate important roles of IFN‐γ in the expansion of CD4+ TCRVβ11+ T cells, and will provide useful information for delineating the regulatory mechanisms of Th1‐cell development and for analyzing mechanisms on Th1‐dominant immune responses.