We investigated how Mycobacterium tuberculosis responded to a reduced oxygen tension in terms of its pathogenicity and gene expression by growing cells under either aerobic or low-oxygen conditions in chemostat culture. The chemostat enabled us to control and vary the oxygen tension independently of other environmental parameters, so that true cause-and-effect relationships of reduced oxygen availability could be established. Cells grown under low oxygen were more pathogenic for guinea pigs than those grown aerobically. The effect of reduced oxygen on global gene expression was determined using DNA microarray. Spearman rank correlation confirmed that microarray expression profiles were highly reproducible between repeat cultures. Using microarray analysis we have identified genes that respond to a low-oxygen environment without the influence of other parameters such as nutrient depletion. Some of these genes appear to be involved in the biosynthesis of cell wall precursors and their induction may have contributed to increased infectivity in the guinea pig. This study has shown that a combination of chemostat culture and microarray presents a biologically robust and statistically reliable experimental approach for studying the effect of relevant and specific environmental stimuli on mycobacterial virulence and gene expression.