The effects of high temperature on multiple parameters of broilers were evaluated both in vitro and in vivo. In the in vitro study, the bilateral musculus fibularis longus muscles of 8 broilers were isolated and incubated at either 41.5 or 44.5 degrees C. The greater incubation temperature increased mitochondrial H2O2 production by 28.0% (P < 0.0001), malondialdehyde concentration by 16.8% (P = 0.0368), and lactate concentration by 33.0% (P < 0.0001) and decreased mitochondrial Ca2+-ATPase activity by 19.6% (P = 0.0001). In the in vivo study, 180 four-week-old broilers were kept in 3 controlled-environment chambers for 3 wk. High temperature increased mitochondrial H2O2 production (P < 0.05) in liver, malondialdehyde concentration in liver and breast muscle, and lactate concentration in breast muscle (P < 0.05). In addition, it inhibited mitochondrial Ca2+-ATPase activity in muscle and liver (P < 0.05). High temperature also significantly decreased initial pH and increased L*, drip loss, and shear force of broiler breast muscle.