Ethylenediamino-di-(0-hydroxyphenylacetic acid) (EDDA) was found to be a suitable iron chelator for investigating mycobactin production in mycobacteria. The optimal conditions of EDDA concentration and time of exposure were established and were used to develop a two-step method for mycobactin production. Applied to representative strains of selected species, the method was found to yield reliable results useful for identification and speciation of mycobacteria.