Increased hydrogen peroxide (H 2 O 2 ) production was observed in duodenal mitochondria obtained from broiler chickens with low feed efficiency (FE). As a decrease in mitochondrial membrane potential (▵ψ m ) due to mild uncoupling of oxidative phosphorylation reduces reactive oxygen species production, this study was conducted to evaluate the effect of uncoupling on ▵ψ m and H 2 O 2 production in duodenal mitochondria isolated from broilers with low (0.48 ± 0.02) and high (0.68 ± 0.01) FE. Membrane potential and H 2 O 2 production were measured fluorometrically and in the presence of different levels of an uncoupler, carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP). The ▵ψ m was higher (P ≤ 0.05) in high FE mitochondria at 0 to 600 nM FCCP. A decrease in ▵ψ m was observed at 600 and 1000 nM FCCP in the low and high FE groups, respectively. H 2 O 2 generation was higher in the low FE mitochondria at all FCCP levels except at 200 nM. Adding 200 to 800 nM FCCP decreased H 2 O 2 production in low but not in high FE mitochondria. These results showed that FCCP-induced uncoupling lowered H 2 O 2 production in low FE but not in high FE duodenal mitochondria and suggest that ▵ψ m may influence H 2 O 2 production in low FE mitochondria.