Mechanisms for the loss of muscle contractile function in hyperthermia are poorly understood. This study identified the critical temperature resulting in a loss of contractile function in isolated diaphragm (thermal tolerance) and then tested the hypotheses (i) that increased reactive oxygen species (ROS) production contributes to the loss of contractile function at this temperature, and (ii) eicosanoid metabolism plays an important role in preservation of contractile function in hyperthermia. Contractile function and passive force were measured in rat diaphragm bundles during and after 30 min of exposure to 40, 41, 42 or 43°C. Between 40-42°C there were no effects of hyperthermia, but at 43°C, a significant loss of active force and an increase in passive force were observed. Inhibition of ROS with the antioxidants, Tiron or Trolox did not inhibit the loss of contractile force at 43°C. Furthermore, treatment with dithiothreitol, a -SH reducing agent, did not reverse the effects of hyperthermia. A variety of global lipoxygenase (LOX) inhibitors further depressed force during 43°C and caused a significant loss of thermal tolerance at 42°C. Cyclooxygenase (COX) inhibitors also caused a loss of thermal tolerance at 42°C. Blockage of phospholipase with PLA2 inhibitors, bromoenol lactone (BEL) or AACOCF3 failed to significantly prevent the loss of force at 43°C. Overall, these data suggest that ROS do not play an apparent role in the loss of contractile function during severe hyperthermia in diaphragm. However, functional LOX and COX enzyme activities appear to be necessary for maintaining normal force production in hyperthermia.