The bloodfed midgut of sand fly vectors is a potentially lethal environment for Leishmania due to the release of proteolytic enzymes involved in bloodmeal digestion. Acquisition of the LPG coat on promastigotes protects these cells from proteolytic attack since promastigote mutants lacking surface LPG were highly susceptible to killing within the bloodfed midgut, whereas transfection of these mutants with genes capable of restoring LPG expression rescued their early midgut survival. Formation of a peritrophic membrane (PM) around the infected bloodmeal appeared to protect transforming parasites from induced proteases since disruption of the PM with exogenous chitinase resulted in almost complete and early loss of infection in these flies. A novel role for the PM in vector competence is suggested, in which containment of the infected bloodmeal within the intra-peritrophic space creates a barrier to the rapid diffusion of gut proteases, and limits the accessibility of these enzymes to the vulnerable forms of parasites in life-cycle transition, prior to their expression of LPG. LPG minus mutants and their rescued transfectants were also used to confirm the role of LPG in midgut attachment. Each of three L. donovani mutants examined failed to attach to P. argentipes midguts in vitro, and failed to maintain infection in the fly after passage of the digested bloodmeal. One transfectant, for which LPG expression had been restored to near normal levels, displayed restored capacity for midgut adhesion both in vitro and in vivo. In contrast, functional analysis of GP63- deficient mutants failed to reveal any role for this abundant surface protease in the life-cycle of the parasite in the fly. No effect of the null mutation was observed on the survival, transformation, growth, and differentiation of parasites in the vector midgut. With respect to the vertebrate host, null mutants were able to attach to and survive within mouse peritoneal macrophages in vitro, and to produce progressive cutaneous lesions in BALB/c mice. Null mutants were, however, significantly less resistant to complement-mediated lysis, and their ability to initiate lesion development was delayed. Studies in the next year will focus on characterization of midgut receptors for LPG using a combination of biochemical and molecular cloning techniques.