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The steroidogenic acute regulatory (StAR)-related lipid transfer (START) domains are found in a wide range of proteins involved in intracellular trafficking of cholesterol and other lipids. Among the START proteins are the StAR protein itself (STARD1) and the closely related MLN64 protein (STARD3), which both function in cholesterol movement. We compared the cholesterol-binding properties of these two START domain proteins. Cholesterol stabilized STARD3-START against trypsin-catalyzed degradation, whereas cholesterol had no protective effect on STARD1-START. [3H]Azocholestanol predominantly labeled a 6.2 kDa fragment of STARD1-START comprising amino acids 83–140, which contains residues proposed to interact with cholesterol in a hydrophobic cavity. Photoaffinity labeling studies suggest that cholesterol preferentially interacts with one side wall of this cavity. In contrast, [3H]azocholestanol was distributed more or less equally among the polypeptides of STARD3-START. Overall, our results provide evidence for differential cholesterol binding of the two most closely related START domain proteins STARD1 and STARD3.