Glutathione S-transferases (GSTs, EC 2.5.1.18) were isolated from the liver cytosolic fraction of 1 day old Leghorn chicks by S-hexylglutathione and glutathione affinity columns arranged in tandem. After sample loading, the affinity columns were detached from each other and developed separately. Four groups of GSTs (CL 1, 2, 3, and 4) were eluted from the hexylglutathione column, and an additional group of GSTs (CL 2 and 5) was eluted from the glutathione affinity column. CL 2, CL 3, and CL 5 were further purified to homogeneity by chromatofocusing, and the substrate specificities of each group were determined. Fractions from the chromatofocusing column were analyzed by native IEF electrophoresis. Protein bands were electroblotted onto PVDF membrane for N-terminal sequence analysis or extracted from IEF gel and rerun on SDS-PAGE to determine the subunit composition of each GST dimer. CL 2, CL 3, and CL 5 can form homodimers, whereas CL 1 and CL 4 exist only as CL 1-2 and CL 3-4 heterodimers. CL 2 and CL 5 have N-terminal amino acid sequences homologous to rat liver Yb and Ya GSTs, respectively. CL 1 has a unique N-terminal sequence that is not homologous to any known GSTs.