Summary Plasma membrane organization exerts tremendous influence on cellular functionality. A well known mechanism underlying this organization is through nanoscopic clustering of distinct lipids and proteins in membrane rafts. Raft domains are enriched in cholesterol and lipids with saturated acyl chains and share properties with liquid-ordered membrane phases observed in vitro. In cell membranes, raft domains are thought to co-exist with more fluid, disordered domains, and the cholesterol-rich environment of the plasma membrane is thought to be especially conducive for raft formation. Individual membrane proteins tend to prefer either raft or non-raft environments, and this propensity serves as an important regulatory mechanism of their function. Further, raft-dependent processes have been implicated in many human pathologies including several forms of cancer, AIDS and Alzheimer's disease. However, the size and dynamics of these domains and how the partitioning of membrane proteins between ordered and disordered domains affect their functions and interactions remain uncertain. We argue that these fundamental gaps in knowledge remain because few methods exist to experimentally perturb rafts in a controlled manner. The objective of this proposal is therefore to discover and validate first generation small molecules that can be used to pharmacologically manipulate rafts. To tackle this problem, we have developed a novel high throughput screen (HTS) assay exploiting giant plasma membrane vesicles (GPMVs) as a model system to visualize rafts and their associated proteins, as well as custom software to quantitate these datasets. Using these approaches, we have now identified several examples of bioactive lipids and FDA-approved drugs that alter the relative proportions of ordered and disordered phases in GPMVs, as well as have preliminary evidence in hand that suggests it is possible to alter the phase partitioning of selected membrane proteins. Here, we propose to build on these initial successes to move from proof-of-principle stage to the point where we have in hand validated first generation small molecules with these activities. Protein targets will include peripheral myelin protein 22, a protein associated with Charcot-Marie-Tooth syndrome, and the HIV receptor CD4 and its co-receptor CCR5. Ultimately, the results of these studies will lay the groundwork for the development of a new class of chemicals that can be used to pharmacologically manipulate rafts in the laboratory and definitively test long-standing questions about the nature of rafts as well as for employment in drug discovery programs.