After infection with Mycobacterium tuberculosis, clinical disease usually remains latent, contained by the host immune response. Although polymorphisms of HLA loci have been hypothesized to play a major role in the breakdown of latency, a functional link has not been established. Molecular-based HLA-typing methods were used to test the association of sets of HLA alleles encoding an aspartic acid at codon 57 of the HLA-DQ beta-chain (HLA-DQ beta57-Asp) with susceptibility to tuberculosis in a cohort of 436 pulmonary tuberculosis patients and 107 healthy controls from Cambodia. HLA class II null cells were transduced with HLA-DQ beta57-Asp or HLA-DQ beta57-Ala and evaluated for their ability to bind peptides from two immunogenic M. tuberculosis specific proteins, ESAT-6 and CFP-10. In this study, we report a highly significant association between progressive pulmonary tuberculosis and homozygosity for HLA-DQ beta57-Asp alleles. The presence of HLA-DQ beta57-Asp resulted in a significantly reduced ability to bind a peptide from the central region of the ESAT-6 protein. Furthermore, when this peptide was presented by an HLA-DQ beta57-Asp allele, Ag-specific IFN-gamma production from CD4+ T cells from tuberculosis patients was significantly less than when this peptide was presented by an HLA-DQ-beta allele encoding an alanine at codon 57. Multiple genetic loci and ethnic-specific factors are likely involved in the human immune response to tuberculosis. The data presented here provide a functional explanation for a highly significant association between an HLA polymorphism and tuberculosis in a highly characterized group of patients with susceptibility to progressive tuberculosis infection in Cambodia.