Mycobacterial growth inhibitory factor (MycoIF), found in supernatant fluids of mouse spleen cell cultures that have been stimulated in vitro with homologous antigen, inhibited the intracellular multiplication of virulent tubercle bacilli within normal mouse peritoneal macrophages in vitro. Antigenically stimulated H37Ra-immunized mouse spleen cells required 72 h of incubation to produce supernatant fluids that would cause intracellular inhibition. Supernatant fluids from 48-h mouse spleen cell cultures were not able to produce intracellular inhibition. Investigation of the culture conditions showed that at lease 1.0% human serum was required in the tissue culture medium for the production of MycoIF by spleen cells from immunized mice. MycoIF activity was noted only in supernatant fluids from spleen cell cultures incubated with antigen for 72 h. MycoIF was nondialyzable and unaffected by freezing, lyophilization, or incubation at 60 C for 30 min. However, MycoIF was inactivated after incubation at 80 C for 30 min. MycoIF was unaffected by low hydrogen ion concentrations (pH 7 to 12), but exposure to higher hydrogen ion concentrations (pH 6, pH 5) significantly decrease MycoIF activity, and exposure to pH 4 to 2 abolished all activity. Supernatant fluids diluted 1:32 were still able to produce significant intracellular inhibition of growth of virulent tubercle bacilli.