Humans infected with the dimorphic fungus Blastomyces dermatitidis develop strong T-lymphocyte responses to WI-1, an immunodominant antigen that has been shown to elicit protective immunity in mice. In the present study, the T-cell epitopes of WI-1 and human leukocyte antigen (HLA) restricting elements that display them were investigated. Peripheral blood mononuclear cells (PBMC) from 37 patients with a confirmed history of blastomycosis were tested for a response to WI-1 in primary proliferation assays; PBMC from 35 (95%) responded. Six patients whose PBMC proliferated strongly in response to WI-1 (defined as a stimulation index greater than 50) were tested further for responses to subcloned, recombinant fragments of the antigen. These patients responded chiefly to sequences within the N terminus and the 25-amino-acid tandem repeat. Cloned CD4⁺ T cells from an infected individual were used to delineate more precisely the peptide epitopes in the fragments and HLA restricting elements that present them. A majority of the T-cell clones recognized an epitope spanning amino acids 149 to 172 within the N terminus, displayed by HLA-DR 15. A minority of the clones, which have been shown to perform a cytolytic function in vitro, recognized an epitope in the tandem repeat displayed by HLA-DPw4, an uncommon restricting element. Tandem repeat epitopes required display by the  chain of DPw4 heterodimers. Thus, human T cells with different functions in vitro also recognize distinct regions of WI-1, raising the possibility that HLA restricting elements that present them could modulate immunity during blastomycosis by selection and display of WI-1 peptides.