Iron, a key nutritional requirement for the in vitro and in vivo multiplication of mycobacteria, regulates the expression of genes involved in its acquisition as well as those encoding cytoplasmic or surface proteins coordinately regulated with components of the iron-sequestration machinery. Mycobactin is an iron chelator produced by most species of mycobacteria with M. paratuberculosis being a notable exception. In our studies, we carried out two-dimensional gel electrophoresis to analyze the effect of varying cultural conditions and iron concentrations on M. avium and M. paratuberculosis protein expression. Approximately 150 polypeptide spots were resolved for cellular and cell-associated proteins. M. avium grown on minimal and Middlebrook 7H9 medium revealed unique polypeptides associated with mycobactin synthesis, low and high iron concentration, and conditions inducing ultrastructural changes. M. paratuberculosis cells grown on Middlebrook 7H9 medium containing either high iron, low iron or ferric mycobactin revealed fewer differences. Two polypeptides of 15 and 70 kDa were predominantly synthesized in high iron medium, while a 23 kDa polypeptide was overproduced under low iron conditions. In conclusion, our studies indicate a differential response of M. avium and M. paratuberculosis gene expression to environments with low and high iron.