Dipeptidase activity in Rhynchosciara americana (Diptera: Sciaridae) is found mainly in midgut caeca cells. The caecal dipeptidase activity is partly soluble and partly membrane bound. Differential centrifugation of midgut caeca homogenates, followed by assays of enzyme markers and dipeptidase, suggest that soluble dipeptidase is cytosolic or weakly associated with the cell glycocalyx. Membrane-bound dipeptidase is likely a microvillar enzyme. Soluble dipeptidase activity is resolved by gel filtration and ion exchange chromatography into two enzymes (M r 63,000 and 73,000), which hydrolyze both Gly-Leu and Pro-Gly, although with different efficiency. The two enzymes also differ in their stability in the presence of EDTA and degree of inhibition by phenanthroline and aminoacyl hydroxamates. Dipeptidase inhibition by phenanthroline is reversed by dialysis. Membrane-bound dipeptidase activity was solubilized by Triton X-100 and papain. Density-gradient ultracentrifugation, gel filtration, and ion-exchange chromatography suggest that there is only one detergent (M r 86,000) form of this enzyme, which is active on Gly-Leu and Pro-Gly. No activity upon Gly-Pro was found in R. americana midguts, whereas the weak activity observed upon carnosine is independent of the enzymes hydrolyzing Gly-Leu and Pro-Gly. Thus, R. americana midguts seem to have major soluble and membrane-bound dipeptide hydrolases (EC 3.4.13.11), which in contrast to the mammalian enzyme, are very active upon Pro-Gly. R. americana also has a minor carnosinase (EC 3.4.13.3).