Three β-glycosidases, named βGly1, βGly2 and βGly3, were isolated from midgut tissues of the sugar cane borer, Diatraea saccharalis Fabricius (Lepidoptera: Pyralidae). The three enzymes have similar Mr (58,000; 61,000; 61,000), pI (7.5, 7.4, and 7.4) and optimum pH (6.7, 6.3, and 7.2) and were resolved by hydrophobic chromatography. The β-glycosidases prefer β-glucosides to β-galactosides, have four subsites for glucose binding and hydrolyse glucose-glucose β-1,3 linkages better than β-1, 4- or β-1,6 linkages. βGly1 and 2 were completely purified, whereas βGly3 was isolated with a contaminant peptide that has no activity upon β-glycosides. By using competing substrates, it was shown that βGly 1 and 3 have one active site, whereas βGly2 has two, one hydrolyzing natural and the other synthetic substrates. βGly2 is the only D. saccharalis β-glycosidase that can efficiently hydrolyse prunasin, the glycoside remaining after glucose removal from the plant glycoside amygdalin and that liberates the cyanogenic mandelonitrile. As shown elsewhere, βGly2 activity is reduced when D. saccharalis is reared in amygdalin containing diets. From the results, we propose that the physiological role of βGly 1 and 3 is the digestion of oligo- and disaccharides derived from hemicelluloses and of βGly2 is glycolipid hydrolysis. Free energy relationships showed that D. saccharalis βGly3 and Tenebrio molitor (Coleoptera) βGly1 have active sites that bind similarly the transition states formed with different substrates. The same is also true for the active sites of D. saccharalis βGly1 and T. molitor βGly2. This suggests that active sites of similar enzymes are probably homologous, displaying nearly identical bonds between active site amino acids and substrate moieties.