Formation of reactive oxygen species (ROS) in mitochondrial isolates from gill tissues of the Antarctic polar bivalve Laternula elliptica was measured fluorimetrically under in vitro conditions. When compared to the rates measured at habitat temperature (1°C), significantly elevated ROS formation was found under temperature stress of 7°C and higher. ROS formation correlated significantly with oxygen consumption in individual mitochondrial preparations over the entire range of experimental temperatures (1-12°C). ROS generation per mg of mitochondrial protein was significantly higher in state 3 at maximal respiration and coupling to energy conservation, than in state 4+, where ATPase-activity is inhibited by oligomycin and only proton leakage is driving the residual oxygen consumption. The percent conversion of oxygen to the membrane permeant hydrogen peroxide amounted to 3.7% (state 3) and 6.5% (state 4+) at habitat temperature (1°C), and to 7% (state 3) and 7.6% (state 4+) under experimental warming to 7°C. This is high compared to 1-3% oxygen to ROS conversion in mammalian mitochondrial isolates and speaks for a comparatively low control of toxic oxygen formation in mitochondria of the polar bivalve. However, low metabolic rates at cold Antarctic temperatures keep absolute rates of mitochondrial ROS production low and control oxidative stress at habitat temperatures. Mitochondrial coupling started to fall beyond 3°C, closely to pejus temperature (4°C) of the bivalve. Accordingly, the proportion of state 4 respiration increased from below 30% at 1°C to over 50% of total oxygen consumption at 7°C, entailing reduced ADP/O ratios under experimental warming. Progressive mitochondrial uncoupling and formation of hazardous ROS contribute to bias mitochondrial functioning under temperature stress in vitro. Deduced from a pejus temperature, heat stress commences already at 5°C, and is linked to progressive loss of phosphorylation efficiency, increased mitochondrial oxygen demand and elevated oxidative stress above pejus temperatures. © 2002 Elsevier Science Inc. All rights reserved.