Most of the carbon monoxide (CO) produced by mammals is a product of the heme oxygenase (HO) reaction, the rate-limiting step in the heme degradation pathway leading to the generation of bilirubin in man. However, some CO is derived from other sources. We studied the association of CO production with lipid peroxidation in tissue preparations from adult male Wistar rats. Supernatants, from 20% tissue homogenates in potassium phosphate buffer, centrifuged for 1 min at 13 000 × g, were incubated for 30 min at 37°C in septum-sealed vials in the dark with ascorbate (100 µM) and Fe(II) (6 µM) and (or) Fe(III) (60 µM). Butylated hydroxytoluene (BHT, 100 µM) was added for the blank reaction. CO produced into the headspace was quantitated by gas chromatography. Thiobarbituric acid reactive substances (TBARS), conjugated dienes (CD), and lipid hydroperoxides (LOOH) in the reaction medium were quantitated by spectrophotometry. Of the tissues studied, CO and TBARS formation was greatest for brain, followed by kidney, lung, spleen, and blood, but no CO or TBARS formation was detected for testes, intestine, liver, and heart. Cell fractionation studies indicated that these differences might be due to the presence of endogenous soluble antioxidants in the latter tissues. Furthermore, these studies demonstrated that CO was exclusively generated by subcellular fractions that contained membranes. The magnitude of the rate of product formation in brain supernatants depended on the concentration of Fe(II) and (or) Fe(III). The formation of CO, TBARS, CD, and LOOH increased linearly with time for up to 30 min, but the rates of product formation were different. Product formation was completely inhibited by BHT (100 µM), biliverdin (50 µM), bilirubin (50 µM), citrate (100 µM), and the Fe(II) chelators, desferrioxamine mesylate (100 µM) and diethylenetriaminepentaacetate, but not by 10 µM of the HO inhibitor, zinc deuteroporphyrin bis glycol. We conclude that CO generation is associated with the process of in vitro lipid peroxidation in tissues with limited antioxidant reserves.Key words: brain, carbon monoxide, conjugated dienes, lipid peroxidation, thiobarbituric acid reactive substances (TBARS).