In Saccharomyces cerevisiae, the rate of hydrogen peroxide (H 2O 2) diffusion through the plasma membrane decreases during adaptation to H 2O 2 by a still unknown mechanism. Here, adaptation to H 2O 2 was observed to modulate rapidly the expression of genes coding for enzymes involved in ergosterol and lipid metabolism. Adaptation to H 2O 2 also alters plasma membrane lipid composition. The main changes were the following: (a) there was a decrease in oleic acid (30%) 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 80% decrease in 2-hydroxy-C26:0 levels and a 50% 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, H 2O 2 leads to a reorganization of the plasma membrane microdomains, which may explain the lower permeability to H 2O 2, and emerges as an important regulator of lipid metabolism and plasma membrane lipid composition.