A phenotypical analysis carried out by indirect immunofluorescence and two-color cytofluorometry showed that the number of lymphocytes bearing the gamma delta T cell receptor (TcR) heterodimer was dramatically increased in the blood of six children with Brucella melitensis infection. Most in vivo expanded gamma delta T cells reacted with a monoclonal antibody which identifies V delta 2 gene products and a significant proportion expressed CD25 and HLA-DR activation antigens. In addition, whereas only a few gamma delta T lymphocytes were CD8+, nearly all were CD4-. Highly enriched populations of both alpha beta and gamma delta T cells were obtained by negative immunoselection from three subjects with brucellosis sampled during convalescence. Despite the different form of their TcR, the proliferation of these two major T cell subsets in response to a mitogenic anti-CD3 monoclonal reagent (OKT3) was optimal. In contrast, alpha beta, but not gamma delta, T lymphocytes proliferated vigorously in response to the antigenic stimulus elicited by heat-killed Brucella. Further studies are, therefore, needed to determine whether the selective expansion of the gamma delta T cell subpopulation observed during the clinical course of the infection is driven by antigenic determinant(s) borne by the pathogen in vivo or is due to host-derived stimuli, such as autologous heat-shock proteins expressed on the surface of the infected cells.