1. A comparative study was conducted on the molecular weights of glutathione S-transferases in the housefly and liver of the mouse and rat using Sephadex G-100 gel chromatography. 2. The values varied depending upon the buffers used in gel filtration. Molecular weights of 44,600, 53,600 and 43,000 daltons respectively were obtained with 0.01 M potassium phosphate buffer, pH 6.7; 0.05 M Tris-HCl buffer, pH 8.0; and 0.05 M Tris-HCl buffer containing 0.1 M KCl, pH 8.0, respectively. 3. There was no difference in the molecular weights of the enzymes obtained from the insect and from the mammalian livers. Purified enzymes eluted in the same fractions as those from the crude extracts, suggesting little modification in the molecular size of the enzymes during purification. 4. The presence of a large volume of stabilizer(s) in the enzyme solutions applied to the column delayed the elution of the activity peaks and resulted in erroneous values. Therefore, different literature values of molecular weights for glutathione S-transferases may be the result of different buffers and stabilizers used in gel filtration and probably do not represent a real difference in molecular size.