Carboxymycobactin, in which the usual intracellular mycobactin siderophore is modified by possession of a carboxylic acid group, has been isolated as a second extracellular siderophore from culture filtrates of Mycobacterium smegmatis grown under iron-deficient conditions. (The primary siderophore is an exochelin which is a trihydroxamate, pentapeptide derivative). There may be up to 12 similar molecules produced with differing chain lengths that can be recognized by HPLC or HPTLC. The amount of carboxymycobactin is about 20 times higher when cultures are grown with glycerol instead of glucose. Formation is maximal with an initial pH of the medium of about 8.4. The proportion of carboxymycobactin to the total siderophore produced--mainly exochelins--is maximally 10% (usually 10-25 micrograms ml(-1)). Formation of both extracellular siderophores (exochelin and carboxymycobactin) and of the intracellular mycobactin is maximal at the same initial concentration of iron added to the medium, 0.05-0.1 micrograms Fe ml(-1), though exochelin is synthesized 24 h in advance of both carboxymycobactin and mycobactin.