Parasites Toxoplasma gondii and Plasmodium, the agent of malaria, are deadly human pathogens. These obligate intracellular parasites must invade host cells to survive, making understanding the parasite invasion machinery an important goal. At the heart of this machinery are rhomboid proteases, which catalyze the essential cleavage of parasite adhesin proteins that are required for attachment to host cells. Rhomboids are integral membrane proteins that cross the membrane seven times, and we previously deduced that they function as novel proteases;their transmembrane domains (TMDs) associate to form a serine protease active site within the membrane bilayer. Remarkably, cleavage of adhesins occurs within their TMDs. Such hydrolysis of peptide bonds within the normally hydrophobic environment of the membrane is a new paradigm in enzyme biochemistry. This paradigm is of wider importance to human health as various intramembrane proteases have recently been implicated as central players in Alzheimers Disease, hypercholesterolemia, and infection by pathogenic bacteria. However, the biochemical function of these unusual membrane enzymes is poorly understood. We seek to decipher how these enigmatic proteases function at the molecular level, with particular emphasis on their role in parasite invasion. Specifically, capitalizing on new biochemical methods for studying rhomboids that we have recently developed, we propose to investigate the following key issues: 1) physical basis of rhomboid substrate specificity compared to that of other intramembrane proteases, 2) arrangement and regulation of rhomboids in parasite membranes, 3) structural arrangement and function of rhomboid proteases, 4) development of small molecule inhibitors of rhomboid catalysis.