The activities of some commonly used antituberculosis drugs were investigated within unstimulated peritoneal macrophages and in 7H-9 medium without Tween 80 using Mycobacterium microti OV254 as the target organism. In macrophage cultures, serial concentrations of isoniazid, rifampin, pyrazinamide, or streptomycin were added after a 2.5-h phagocytosis period. Viable counts were carried out at daily intervals for 5 or 6 days. The patterns of susceptibility to the four drugs were similar for M. microti and Mycobacterium tuberculosis. To ensure comparability with daily drug replacements in the macrophage experiments, the period of exposure to drugs in 7H-9 medium was kept to only 3 days. With in vitro culture at pH 6.4, drug penetration, measured as the ratio of MICs in macrophages to MICs in 7H-9 medium, was approximately 5 for isoniazid, 5 for rifampin, and 10 for streptomycin. With in vitro culture at pH 7.4, drug penetration was 100 for streptomycin, and at pH 5.6 it was 1 for pyrazinamide. Pyrazinamide was only bacteriostatic in macrophages but weakly bactericidal during the first day of exposure in vitro.