Paracoccidioides brasiliensis, a thermal dimorphic fungus, is the etiologic agent of the most common systemic mycosis in Latin America, paracoccidioidomycosis. The yeast form of P. brasiliensis acts as a facultative intracellular pathogen being able to survive and replicate within the phagosome of nonactivated murine and human macrophages. This ability has been proposed to be crucial to the development of disease. Thus, P. brasiliensis may have evolved mechanisms that counteract the constraints imposed by phagocytic cells. By using cDNA microarray technology we evaluated the early transcriptional response of this fungus to the environment of peritoneal murine macrophages in order to shed light on the mechanisms used by P. brasiliensis to survive within phagocytic cells. Of the 1152 genes analyzed, we identified 152 genes that were differentially transcribed. Intracellularly expressed genes were primarily associated with glucose and amino acid limitation, cell wall construction, and oxidative stress. For the first time, a comprehensive gene expression tool is used for the expression analysis of P. brasiliensis genes when interacting with macrophages. Overall, our data show a transcriptional plasticity of P. brasiliensis in response to the harsh environment of macrophages which may lead to adaptation and consequent survival of this pathogen.