Two Spodoptera litura glutathione S-transferase cDNAs (Slgste2 and Slgste3) which were cloned from a midgut cDNA, encoded two structurally distinct proteins with a predicted molecular mass of 25 and 24kDa, respectively. Slgste2 and Slgste3 were single-copy genes in the S. litura genome and there was no intron within the genes. The transcripts and proteins of Slgste2 and Slgste3 were predominately present in the midgut of the 5th and 6th instar larvae. The apparent Vmax of the purified SlGSTE2 and SlGSTE3 recombinant proteins towards the substrates glutathione and 1-chloro-2,4-dinitrobenezene (CDNB) were similar. Slgste2 expression was up-regulated by 1-naphthyl methylcarbamate (carbaryl), 1,1,1-trichloro-2,2-bis-(p-chlorophenyl) ethane (DDT), deltamethrin, tebufenozide (RH5992) and Bacillus thuringiensis (Bt), but not affected by malathion, while Slgste3 expression was slightly up-regulated by carbaryl, Bt and DDT, but not affected by RH5992, malathion and deltamethrin. The results suggest that Slgste2 and Slgste3 may play roles in detoxifying various insecticides in S. litura.