Steroidogenic acute regulatory protein (StAR) and peripheral-type benzodiazepine receptor (PBR) have both been implicated in the transport of cholesterol across mitochondrial membranes in steroidogenic cells. Therefore, we hypothesized that StAR and PBR were associated in this process. To test this hypothesis, we measured fluorescence energy transfer (FET) between these proteins by fusing enhanced green fluorescent protein (GFP, donor fluorophore) and yellow fluorescent protein (YFP, acceptor fluorophore) to the C-terminus of ovine StAR (37GFP) and ovine PBR (PBRYFP), respectively. These intrinsically fluorescent proteins were stably transfected into Cos-7 cells and determined to be biologically active. For FET to occur the appropriate fluorescent molecules need to be <100 Å from each other. We observed 22.0 ± 0.9% energy transfer efficiency for 37GFP and PBRYFP, a 4.9 fold increase above non-specific energy transfer between free GFP and PBRYFP (p < .0001). Thus, it appears that StAR and PBR are closely associated in mitochondrial membranes and that these molecules may interact in the transportation of cholesterol.