The molecular mechanisms by which plants acclimate to oxidative stress are poorly understood. To identify the processes involved in acclimation, we performed a comprehensive analysis of gene expression in Nicotiana tabacum leaves acclimated to oxidative stress. Combining mRNA differential display and cDNA array analysis, we estimated that at least 95 genes alter their expression in tobacco leaves acclimated to oxidative stress, of which 83% are induced and 17% repressed. Sequence analysis of 53 sequence tags revealed that, in addition to antioxidant genes, genes implicated in abiotic and biotic stress defenses, cellular protection and detoxification, energy and carbohydrate metabolism, de novo protein synthesis, and signal transduction showed altered expression. Expression of most of the genes was enhanced, except for genes associated with photosynthesis and light-regulated processes that were repressed. During acclimation, two distinct groups of coregulated genes ("early-" and "late-response" gene regulons) were observed, indicating the presence of at least two different gene induction pathways. These two gene regulons also showed differential expression patterns on an oxidative stress challenge. Expression of "late-response" genes was augmented in the acclimated leaf tissues, whereas expression of "early-response" genes was not. Together, our data suggest that acclimation to oxidative stress is a highly complex process associated with broad gene expression adjustments. Moreover, our data indicate that in addition to defense gene induction, sensitization of plants for potentiated gene expression might be an important factor in oxidative stress acclimation.