Pathogenetic processes that facilitate the entry, replication, and persistence of Mycobacterium tuberculosis (MTB) in the mammalian host likely include the regulated expression of specific sets of genes at different stages of infection. Identification of genes that are differentially expressed in vivo would provide insights into host-pathogen interactions in tuberculosis (TB); this approach might be particularly valuable for the study of human TB, where experimental opportunities are limited. In this study, the levels of selected MTB mRNAs were quantified in vitro in axenic culture, in vivo in the lungs of mice, and in lung specimens obtained from TB patients with active disease. We report the differential expression of MTB mRNAs associated with iron limitation, alternative carbon metabolism, and cellular hypoxia, conditions that are thought to exist within the granulomatous lesions of TB, in the lungs of wild-type C57BL/6 mice as compared with bacteria grown in vitro. Analysis of the same set of mRNAs in lung specimens obtained from TB patients revealed differences in MTB gene expression in humans as compared with mice.