In vivo labeling of proteins using 35S-methionine followed by one or two dimensional analysis is a convenient method for studying the pattern of gene expression in M.ptb and other mycobacteria under a variety of controlled environmental conditions in the laboratory. M.ptb strain Dominic, a human disease isolate of M. avium and M. bovis BCG, were cultured in Dubos medium, supplemented with mycobactin in the case of M.ptb. Organisms in the mid log phase of growth were transferred to RPMI 1640 methionine deficient medium in the presence of 35S-met and incubated for 2h at 37°C or 45°C heat shock. Cells were harvested, washed in Tris buffer and lysed by beating with < 100 mcm glass beads for 5 min. After boiling, proteins were separated on 10% SDS-PAGE, the gels dried and autoradiographed. In other studies the mycobacteria were grown intracellularly in THP-1 human macrophages for 24h prior to 35S-met labeling. After lysis of the macrophages, labeled mycobacteria were harvested by centrifugation and the pattern of 35S-met mycobacterial proteins obtained as described. The profile of labeled proteins from all three mycobacteria in conventional culture and their responses to heat shock were remarkably similar. Substantial differences were however observed in the pattern of labeled proteins when the organisms were cultured in THP-1 macrophages. These methods are applicable to the study of adaptation in mycobacteria in response to the intracellular environment.