The three-layered peritrophic matrix of Glossina morsitans morsitans is shown, by histochemistry, to be formed of a mixture of glycosaminoglycans, glycoproteins and chitin. In all three layers the glycosaminoglycans contain GlcNAc-hexuronic and Gal-GlcNAc moieties, together with chitin. Glycosaminoglycans in layer 3 are sulphated and sulphated sites have a mean interspace distance of 53 nm – similar to the spacing of fixed charge sites in glomerular basement mem- brane suggesting a rôle for these sites in the filtration properties of the peritrophic matrix. O-linked oligo- saccharides are present in all three layers. Layer 1 contains the widest variety of glycoprotein oligosaccharide constructs GlcNac and αlinked GalNAc possibly as GalNAcα1,3GalNAc, the latter apparently distal to Galβ1–4GlcNAc. Lectin binding suggests that layer 2 contains GalNAcα1,3Galβ1,3GlcNAc and that layer 3 contains GalNAc and Galβ1,4GlcNAc. The evidence for N-linked oligosaccharides is more equivocal. Two dimensional electrophoresis showed that the peritrophic matrix contains a range of proteins, most of which require relatively harsh treatment for their solubilization.