There are four beta-glycosidases (betagly1, betagly2, betagly3, and betagly4) in Tenebrio molitor midgut larvae. betagly1 and betagly2 have identical kinetic properties, and differ in a few amino acid residues. Purified betagly1 was used to raise antibodies in a rabbit. The resulting antiserum recognizes in a Western blot only betagly1 and betagly2 in midgut tissue homogenates and contents. An immunocytochemical study carried out using confocal fluorescence and immunogold techniques showed that betagly1+betagly2 are secreted by exocytosis mainly from the distal part of the second third of T. molitor midguts. This is the first immunocytochemical study of an insect digestive enzyme that does not have polymers as substrates. Enzyme assays with 0.3 mM amygdalin, a condition that detects only betagly1+betagly2, revealed that most of those beta-glycosidases are found in the lumen of anterior and middle midgut. This supports the hypothesis that a countercurrent flux of fluid occurs in T. molitor midgut that is able to carry betagly1 and betagly2 to anterior midgut, in agreement with the enzyme recycling mechanism thought to occur in most insects.