Phosphatidylcholine transfer protein (PC-TP, also referred to as StarD2) is a highly specific intracellular lipid-binding protein that catalyzes the transfer of phosphatidylcholines between membranes in vitro. Recent studies have suggested that PC-TP in vivo functions to regulate fatty acid and glucose metabolism, possibly via interactions with selected other proteins. To begin to address the relationship between activity in vitro and biological function, we undertook a high-throughput screen to identify small-molecule inhibitors of the phosphatidylcholine transfer activity of PC-TP. After adapting a fluorescence quench assay to measure phosphatidylcholine transfer activity, we screened 114,752 compounds of a small-molecule library. The high-throughput screen identified 14 potential PC-TP inhibitors. Of these, 6 compounds exhibited characteristics consistent with specific inhibition of PC-TP activity, with IC(50) values that ranged from 4.1 to 95.0muM under conditions of the in vitro assay. These compounds should serve as valuable reagents to elucidate the biological function of PC-TP. Because mice with homozygous disruption of the PC-TP gene (Pctp) are sensitized to insulin action and relatively resistant to the development of atherosclerosis, these inhibitors may also prove to be of value in the management of diabetes and atherosclerotic cardiovascular diseases.