Like most bacteria, the enteric mycobacterial pathogens, M. paratuberculosis and M. avium, require iron for growth and replication. Virtually no iron is available in mammalian tissues as it is complexed by host iron-binding proteins (IBP's) such as transferrin, lactoferrin and ferritin. Most enteric bacteria overcome iron deprivation in the host environment either by synthesizing high affinity iron-chelating/uptake systems or by directly binding specific IPB's. We sought to examine the influence of iron-containing compounds on mycobacterial growth. In a chemically defined medium, growth and replication of clinical isolates of mycobacteria derived from intestinal tissues were shown to be regulated by controlling the amount of iron either in the form of iron salts or bound to specific host IBP's at various levels of saturation. All mycobacterial strains ceased growth under conditions devoid completely of iron. Intracellular growth of mycobacteria, using the J774 murine peritoneal macrophage-like host cell line, was also influenced by iron availability. Mycobacteria-infected macrophages demonstrated increased uptake of ferric-transferrin by immunohistochemical staining which was accompanied by increased burdens of mycobacteria observed visually and confirmed quantitatively. Selected strains were cultivated under conditions of iron-deprivation and iron-repleteness, and cell envelope fraction examined for variations in expression of proteins. Up to 8 different iron-regulated envelope proteins (IREP's) were demonstrated in SDS-PAGE preparations and are being examined further for iron acquisition/transport roles. We conclude that iron plays an important role in intestinal mycobacteriosis and suggest several alternative strategies for inhibiting mycobacterial growth including: 1) iron-witholding or deprivation approaches, 2) use of cytokines to down-regulate transferrin receptor expression on host cells, and 3) use of antibiotics which enter and kill mycobacteria through iron-uptake systems.