Thiobarbituric acid (TBA) assay of rat liver homogenate was performed by four general variations with 0.01% butylated hydroxytoluene. Development of red pigment was greatly dependent on the methods. The pigment formation by each method was dramatically increased by introduction of 2 mM t-butyl hydroperoxide (t-BuOOH). The pH values of the reaction mixtures greatly affected the pigment yield both in the absence and presence of t-BuOOH, and the maximal pigment yield was obtained at pH 3-4. These characteristic profiles of the pigment formation were similar to those from alkadienals and essentially different from those from malonaldehyde. Alkadienals were likely candidates for the TBA-reactive substances in the homogenate.