Malaria parasites (ookinetes) appear to digest the peritrophic membrane in the mosquito midgut during penetration. Previous studies demonstrated that lectins specific for N-acetylglucosamine bind to the peritrophic membrane and proposed that the membrane contains chitin [Rudin, W. & Hecker, H. (1989) Parasitol. Res. 75, 268-2791 In the present study, we show that the peritrophic membrane is digested by Serratia marcescens chitinase (EC 3.2.1.14), leading to the release of N-acetylglucosamine and fragmentation of the membrane. We also report the presence of a malaria parasite chitinase that digests 4-methylumbelliferyl chitotriose. The enzyme is not detectable until 15 hr after zygote formation, the time required for maturation of the parasite from a zygote to an ookinete, the invasive form of the parasite. At 20 hr, the enzyme begins to appear in the culture supernatant. The chitinase extracted from the parasite and found in the culture supernatant consists of a major band and two minor bands of activity on native polyacrylamide gel electrophoresis. The presence of chitin in the peritrophic membrane, the disruption of the peritrophic membrane during invasion, and the presence of chitinase in ookinetes suggest that the chitinase in ookinetes is used in the penetration of the peritrophic membrane.The peritrophic membrane found in the midgut of arthropods is defined as a membranous sac surrounding the ingested blood (1). The function of the peritrophic membrane in the various arthropod orders is unclear, but there is speculation that in bloodsucking insects it is one of the factors determining the competence for the transmission of parasites (2,3). These insects form a peritrophic membrane in response to the mechanical distention resulting from the uptake of blood (4). In Aedes aegypti, formation starts immediately after the blood meal, but about 12 hr elapse before it attains a mature structure (5, 6). Plasmodium gallinaceum, an avian malaria parasite that infects A. aegypti, develops motile ookinetes in the blood meal in 16-20 hr (3, 7). Parasites begin to appear in the midgut epithelial cells around 24 hr, but the major invasion occurs around 30 hr (3). As the peritrophic membrane is fully formed in 12 hr and does not dissolve until 48 hr after the blood meal, the parasite must traverse the peritrophic membrane to move from the blood meal to the epithelial cells. A recent ultrastructural study (3) produced direct evidence that the peritrophic membrane is a barrier to infection. The peritrophic membrane surrounding the penetrating ookinetes lost its laminated structure and was less electron dense. Electron-dense material extended from the anterior end of the parasite. A similar modification was previously observed with Babesia microti passing through the peritrophic membrane of Ixodes ticks (8). Because evidence has been presented that the peritrophic membrane contains chitin (9), we searched for chitinase activity in the parasite that might be used to cross the peritrophic membrane. We report here that P...