The acute steroidogenic response, which produces steroids in response to stress, requires the steroidogenic acute regulatory protein (StAR). StAR, a mitochondrial matrix protein, acts on the outer mitochondrial membrane (OMM) to facilitate the movement of cholesterol from the outer to inner mitochondrial membrane via an unknown mechanism. The N-terminal sequence was reported to be nonessential for activity. We show that alteration of the StAR amino-terminal sequence does not change the thermodynamic stability of StAR but offers protection from proteolytic degradation. A longer association between StAR and the OMM strengthens the interaction with cholesterol. Far-UV CD spectra showed that the smaller fragments of StAR domains were less alpha-helical compared to N-62 StAR but were structured as determined by limited proteolysis followed by mass spectrometry. The START domain consisting of amino acids 63-193 also exhibited protease protection for amino acids 84-193. The Stern-Volmer quenching constant (K(SV)) of the N-62 StAR protein is 12.1 x 10(5) M(-1), with all other START fragments having significantly smaller K(SV) values ranging from 6 to 10 x 10(5) M(-1), showing that N-62 StAR has a more open conformation. Only N-62 StAR protein is stabilized with cholesterol having an increased DeltaH value of -5.6 +/- 0.3 kcal/mol at 37 degrees C. These findings demonstrate a mechanism in which StAR is stabilized at the OMM by cholesterol to initiate its massive import into mitochondria.