Mycobacterium tuberculosis is one of the most prolific pathogens worldwide, and its virulence resides in its capacity to survive in human macrophages. In the present study, we analyzed the gene expression profile of M. tuberculosis H37Rv in macrophages and synthetic medium at the whole genome level. Out of 3875 spots tested, 970 genes passed the statistical significance filter (t scores +/-2.5). A total of 22% of those assayed were found to be active genes (up- or downregulated), representing 5.5% of the whole MTB genome. Interestingly, 32.5% of the genes induced in our macrophage experiments are still classified as hypothetical proteins; 19.5% take part in the cell wall and processes (half of which are membrane proteins); 16% are involved in regulation and information pathways; and the PE family accounts for 3.6% of total induced genes. It is important to note that in the course of MTB replication in macrophages, we observed the upregulation of alternative sigma factor sigG and 13 MTB transcriptional regulators. The data for a selected group of upregulated genes were confirmed by real-time RT-PCR. The global MTB transcriptome described in this study suggests an intracellular MTB actively sensing its environment; it repairs and synthesizes its cell wall and DNA, so as to either repair oxidative and nitrosative damage and/or to augment its copy number and evade host cell killing. As far as we know, this is the first study describing MTB expression profiles using whole genome macroarrays during primary human macrophage infection.