The purpose of this study was to determine the effects of dietary fat quantity and fatty acid composition on hepatic H2O2-metabolizing systems, activities of NADPH-generating enzymes and lipid peroxidation. One-month-old male C57BL/6J mice were fed one of six diets: (i) 5% fat, rich in 18:2n-6 fatty acid (5% N-6); (ii) 20% fat. rich in 18:3n-3 (N-3); (iii) 20% fat, rich in 18:2n-6 (N-6); (iv) 20% fat, rich in 18:1n-9 (N-9); (v) 20% fat, rich in saturated fatty acids (SAT); and (vi) 20% fat, deficient in essential fatty acids (EFAD); for 11 wk. Comparisons between animal groups receiving different fat quantities showed that activities of glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) and malic enzyme (ME, EC 1.1.1.40) and the levels of conjugated dienes were significantly lower in the N-6 than in 5% N-6 group. Conversely, activities of catalase (CAT, EC 1.11.1.6) and selenium-glutathione peroxidase (SeGSHPx, EC 1.11.1.9) were higher in the N-6 than in 5% N-6 group. Among the five dietary groups receiving 20% fat but differing in fatty acid composition, CAT activity was lower in the N-9 group, SeGSHPx activity was lower in the EFAD group, and glutathione reductase (GSSGR, EC 1.6.4.2) activity was higher in the N-6 than in the N-3, N-9, SAT and EFAD groups. The EFAD group had much higher levels of total lipids and conjugated dienes, as well as activities of NADPH-generating enzymes, including G6PDH, ME and isocitrate dehydrogenase (EC 1.1.1.42). than the other four high-fat groups. The hepatic levels of malondialdehyde were not different among the five groups fed 20% fat. In the EFAD group, higher hepatic lipid content can be attributed to higher activities of NADPH-generating enzymes, and the elevation of conjugated diene levels may be related to increased oxygenation of 20:3n-6 (Mead acid) via the lipoxygenase/cyclooxygenase pathway. In short, both dietary fat quantity and fatty acid composition selectively affected hepatic H2O2-metabolizing systems, activities of NADPH-generating enzymes and lipid peroxidation status.