Proteins secreted from Mycobacterium tuberculosis during growth are believed to be important for protective immunity against tuberculosis. We have investigated the growth of M. tuberculosis in an enriched liquid medium. The release of isocitrate dehydrogenase from the bacilli served as a marker of autolysis and was observed during the late logarithmic growth phase. The release of proteins during the culture period was investigated by enzyme-linked immunosorbent assay and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Three major groups of proteins, which differed markedly with respect to profile of release and location in intact bacilli, were defined. A short-term filtrate devoid of autolytic products was defined and found to be composed of 33 major components. Five proteins were identified by monoclonal antibodies. Pronounced superoxide dismutase activity was detected in the filtrate. The enzyme was purified and identified as a dominating component of short-term filtrate.