Studies both on the 2-thiobarbituric acid assay (fat autoxidation, fat rancidity) and 2-thiobarbituric acid-aldehyde reactions were carried out. Physico-chemical properties in solution of the reagents (Zthiobarbituric acid, malonaldehyde) and the red adduct, especially, were evaluated. The 2-thiobarbituric acid structure did not change if heated in diluted acid media (until 1% v/v concentration for strong acids) or in 50% v/v concentration for weak acids, as acetic acid. For high acid concentrations, especially with oxyacids (phosphoric acid, trichloroacetic acid), an oxidant hydrolytical effect can be observed. Diluted solutions of malonaldehyde, in a diluted weak acid medium, remained stable for more than ten days. For the first time the stoichiometries of the red and yellow adducts in solution were determined. Both the absorption maxima and molar absorptivity of the red adduct in acid medium were determined. The neutral and alkaline media were less favourable for the assay sensitivity and stability. The more suitable organic solvents for using in the optimized 2-thiobarbituric acid assay in aqueous medium were cyclohexane, chloroform and the ethers, because the red adduct was not very soluble (less than 0.1 g/l) in the organic solvents (fat extractors).