The TIA-1-interacting protein Fas-activated serine/threonine phosphoprotein (FAST) is a component of a signaling cascade that is initiated by ligation of the Fas receptor. Immunofluorescence microscopy using affinity-purified antibodies raised against recombinant FAST reveals that the endogenous protein associates with mitochondria. Subcellular fractionation confirms that FAST is a component of mitochondria. FAST is tethered to mitochondria by a lysine/arginine-rich domain at its carboxyl terminus that is structurally similar to the mitochondrial tethering motifs of monoamine oxidase B and cytochrome b5. At the mitochondrial membrane, FAST interacts with BCL-X-L. The BCL-XL binding domain maps to a BCL-2-homology-3 (BH3)-related domain that is distinct from the mitochondrial-tethering domain (MTD). Although interactions between FAST and BCL-XL require both the BH3-related domain and the MTD, the requirement for mitochondrial tethering can be conferred by a heterologous MTD. Our results suggest that FAST-BCL-X-L interactions are likely to regulate mitochondrial metabolism during Fas-induced apoptosis. (C) 2004 Elsevier Inc. All rights reserved.