Melioidosis is a potentially fatal disease caused by the bacterium Burkholderia pseudomallei. Individuals with subclinical melioidosis have no apparent clinical signs or symptoms, and are identified only by positive serology. The present study is the first to investigate cell-mediated immune (CMI) responses following in vitro stimulation with B. pseudomallei antigens in peripheral blood mononuclear cells (PBMC), collected under field conditions in Papua New Guinea (PNG) from individuals with exposure to B. pseudomallei (n = 13). While five had a clinical history of melioidosis (C(+)), the remaining individuals (n = 8) were seropositive, yet healthy with no clinical history of melioidosis (S(+)/C(-)). Proliferation and IFN-gamma production were significantly greater in lymphocyte cultures from S(+)/C(-) individuals compared to C(+) individuals (P < 0.001 and P < 0.05, respectively). These findings demonstrate that compared to C(+) patients, individuals with subclinical melioidosis have a stronger CMI response to B. pseudomallei antigens in vitro. Such a response may be essential for protection against disease progression.