Most T-cell epitopes are recognized in the context of a single or limited number of major histocompatibility complex (MHC) class II molecules. We have shown previously, however, that the immunodominant p61-80 epitope from the Mycobacterium tuberculosis 19,000 MW protein is recognized in a genetically permissive manner. In this study, permissive recognition of p61-80 was analysed in three murine MHC haplotypes (H-2b,d and k) with respect to: (i) T-cell-epitope core structure; (ii) I-A/I-E class II MHC restriction; and (iii) the identification of critical amino acid residues within the core region. Overlapping epitope core sequences composed of 6 to 8 amino acids were identified for each of the three H-2 haplotypes by T-cell epitope scanning (PEPSCAN) using peptide-specific T-cell lines. The epitope core sequences recognized by peptide and 19,000 MW protein-specific T cells were similar. In all three haplotypes, responses to p61-80 were restricted by class II MHC I-A molecules. To identify residues within the epitope core critically required for recognition, single substitution (alanine or leucine) analogue peptides were tested for their capacity to stimulate p61-80-specific T-cell hybridomas. A heterogeneous pattern of reactivity was observed, even among individual hybridomas derived from the same H-2 haplotype. Although every core residue could be defined as critical for at least one hybridoma, only one critical substitution (74Val-->Ala) was common to all hybridomas. The identification and structural analysis of genetically permissive epitopes of mycobacteria may be a useful strategy for the rational design of peptide-based vaccines for tuberculosis.