The effects of Fe3+, lipid peroxy radicals and the antioxidant butylated hydroxytoluene on the 2-thiobarbituric (TBA) acid quantitation of plasma lipid peroxides were investigated. Whole plasma and plasma fractions prepared by trichloroacetic acid (TCA) protein precipitation and lipid extraction, demonstrated markedly differing TBA reactivities in the presence or absence of added Fe3+. Examination of the spectral profiles of the TBA reacted whole plasma and TCA precipitated fractions demonstrated the presence of interfering compounds which gave rise to an artifactual increase in lipid peroxide concentrations. In contrast the TBA reacted lipid extracts had low levels of interfering compounds that could be removed by our previously described high pressure liquid chromatographic method (Wade, Jackson and van Rij (1985) Biochem. Med. 33, 291-296). Further characterization of the TBA reactivity of the lipid extract showed that Fe3+ at an optimal concentration of 0.5 mM was necessary for the quantitative decomposition of the lipid peroxides to the TBA reactive product malondialdehyde (MDA). However the presence of Fe3+ resulted in further peroxidation of any unsaturated lipids present. Butylated hydroxytoluene (BHT) at an optimal concentration of 1.4 mM inhibited Fe3+ stimulated peroxidation without affecting the formation of the MDA-TBA chromogen. Using a standardized TBA test with plasma lipid extracts and the addition of optimal concentrations of Fe3+ and BHT, we have determined the mean concentration of lipid peroxides in 30 healthy human subjects to be 102.7 +/- 20.0 ngm/ml.