Steroidogenic acute regulatory protein (StAR, STARDI) is a key factor of intracellular cholesterol transfer to mitochondria, necessary for adrenal and gonadal steroidogenesis, and is an archetypal member of the START protein family. Despite the common overall structural fold, START members differ in their binding selectivity toward various lipid ligands, but the lack of direct structural information hinders complete understanding of the binding process and cholesterol orientation in the STARDI complex in particular. Cholesterol binding has been widely studied by commercially available fluorescent steroids, but the effect of the fluorescent group position on binding remained underexplored. Here, we dissect STARDI interaction with cholesterol-like steroids bearing 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) group in different positions, namely, with 22-NBD-cholesterol (22NC), 25-NBD-cholesterol (25NC), 20-((NBDamino)-pregn-5-en-3-ol (20NP) and 3-(NBDamino)-cholestane (3NC). While being able to stoichiometrically bind 22NC and 20NP with high fluorescence yield and quantitative exhaustion of fluorescence of some protein tryptophans, STARDI binds 25NC and 3NC with much lower affinity and poor fluorescence response. In contrast to 3NC, binding of 20NP leads to STARD1 stabilization and substantially increases the NBD fluorescence lifetime. Remarkably, in terms of fluorescence response, 20NP slightly outperforms commonly used 22NC and can thus be used for screening of various potential ligands by a competition mechanism in the future. (C) 2018 Elsevier Inc. All rights reserved.