In Saccharomyces cerevisiae, the rate of hydrogen peroxide (H2O2) diffusion through the plasma membrane decreases during adaptation to H2O2 by a still unknown mechanism. Here, adaptation to H2O2 was observed to modulate rapidly the expression of genes coding for enzymes involved in ergosterol and lipid metabolism. Adaptation to H2O2 also alters plasma membrane lipid composition. The main changes were the following: (a) there was a decrease in oleic acid (30k) and in the ratio between unsaturated and saturated long-chain fatty acids; (b) the phosphatidylcholine:phosphatidylethanolamine ratio increased threefold; (c) sterol levels were unaltered but there was an increased heterogeneity of sterol-rich microdomains and increased ordered domains; (d) the levels of the sterol precursor squalene increased twofold, in agreement with ERG1 gene down-regulation; and (e) C26:0 became the major very long chain fatty acid owing to an 80k decrease in 2- hydroxy-C26:0 levels and a 50k decrease in C20:0 levels, probably related to the down-regulation of fatty acid elongation (FAS1, FEN1, SUR4) and ceramide synthase (LIP1, LAC1) genes. Therefore, H2O2 leads to a reorganization of the plasma membrane microdomains, which may explain the lower permeability to H2O2, and emerges as an important regulator of lipid metabolism and plasma membrane lipid composition.