Exposure of mycobacterial growth inhibitory factor (MycoIF) to trypsin, chymotrypsin, or neuraminidase decrease its ability to produce intracellular inhibition of mycobacterial growth within macrophages, suggesting that MycoIF was a glycoprotein. MycoIF was unaffected by deoxyribonuclease or ribonuclease. Supernatant fluids from antigenically stimulated H37Ra-immunized mouse spleen cells exposed to puromycin were unable to produce significant intracellular inhibition. This indicated that the presence of MycoIF activity in supernatant fluids required protein synthesis. The filtration of MycoIF-containing supernatant fluids on Sephadex G-150 demonstrated that significant MycoIF activity appeared only in those fractions which eluted on the downward side of the serum albumin peak. Based on protein standards filtered through the Sephadex gel, the molecular weight of MycoIF was calculated to be between 20,000 and 35,000. These calculations assumed that MycoIF is a globular protein. Attempts to purify MycoIF by anion exchange chromatography (diethylaminoethylcellulose) was not successful.