Butylated hydroxytoluene (BHT) is a phenolic antioxidant used widely in processed foods and petroleum products. BHT tends to accumulate in animal tissues rich in lipids during the daily intake (Daniel and Gage 1965; Gilbert and Golberg 1965). Its accumulation in human adipose tissues was found by colorimetry (Collings and Sharratt 1970) and by gas chromatography with an electron-capture detector (GC-ECD) after being derivatized (Mizutani and Ohe 1976). In a previous study (Mizutani et al. 1983) dealing with the metabolism of BHT in mice, we analyzed 2,6di-tert-butyl-4hydroxy-4-methyl-2,5-cyclohexadienone (4-hydroxy-BHT), a metabolite of BHT, at nanogram levels by GC-ECD. A peak having the same retention time as 4-hydroxy-BHT appeared often on gas chromatograms of the liver and lung extracts from untreated mice. This peak was identified as 4-hydroxy-BHT by thin-layer chromatography, gas chromatography, and gas chromatography-mass spectrometry (GC-MS) with selected ion monitoring. This paper deals with the identification and determination of 4-hydroxy-BHT residues in the tissues of untreated laboratory animals.