Evidence of a direct association between ferri-exochelin, the major extracellular siderophore of Mycobacterium smegmatis, and a 29 kDa protein has been obtained by three separate methods. (1) Direct binding of 55Fe(III)-exochelin by the 29 kDa protein in an envelope preparation from iron-deficient cells was demonstrated by the extraction of a complex with the nondenaturing detergent CHAPS, and subsequent CHAPS-PAGE and autoradiography. (2) Affinity chromatography on a chemically synthesized ferri-exochelin-Sepharose 4B matrix led to the retention of the 29 kDa protein and a 25 kDa protein. The smaller protein was partially eluted with 1 mM ferri-exochelin although it did not form a stable complex with ferri-exochelin. The 29 kDa protein could not be eluted from the affinity matrix with 1 mM ferri-exochelin either alone or with 1 M NaCl. Only 2% (w/v) SDS could do this, but resulted in protein denaturation. (3) Incubation of 55Fe-exochelin with CHAPS-solubilized envelope proteins in free solution followed by ion-exchange chromatography resolved three radioactive peaks; subsequent analysis by SDS-PAGE showed that the peak with the highest 55Fe-binding activity per unit protein contained both the 29 and 25 kDa proteins. A direct association was demonstrated between the 29 kDa protein and 55Fe-exochelin by gel filtration. The evidence suggests that the 29 kDa iron-regulated envelope protein of M. smegmatis is a ferri-exochelin-binding protein and that the 25 kDa protein, which corresponds in size to a previously reported iron-regulated envelope protein in this bacterium, may have a role in the formation or maintenance of this complex. Proteins extracted from the cell envelope of iron-deficient M. smegmatis with CHAPS were dialysed to remove the detergent, incorporated into liposome suspensions and then incubated with 55Fe(III)-exochelin. This increased the retention of 55Fe by 133-fold compared to proteins not placed in liposomes. Retention of 55Fe was dependent upon the protein loading of the liposomes. Gel filtration confirmed that the iron was retained by these vesicles and even after dialysis the majority of 55Fe was still retained by the vesicles. Re-solubilization of the labelled proteo-liposomes in various detergents gave limited recovery of a ferri-exochelin-protein complex. Attempts to resolve this complex by Triton X-100 PAGE led to separation of the two entities. The complex was stable, however, in a CHAPS-PAGE system.