Apoptosis‐inducing factor (AIF) is encoded by one single gene located on the X chromosome. AIF is ubiquitously expressed, both in normal tissues and in a variety of cancer cell lines. The AIF precursor is synthesized in the cytosol and is imported into mitochondria. The mature AIF protein, a flavoprotein (prosthetic group: flavine adenine dinucleotide) with significant homology to plant ascorbate reductases and bacterial NADH oxidases, is normally confined to the mitochondrial intermembrane space. In a variety of different apoptosis‐inducing conditions, AIF translocates through the outer mitochondrial membrane to the cytosol and to the nucleus. Ectopic (extra‐mitochondrial) AIF induces nuclear chromatin condensation, as well as large scale (∼50 kb) DNA fragmentation. Thus, similar to cytochrome c, AIF is a phylogenetically old, bifunctional protein with an electron acceptor/donor (oxidoreductase) function and a second apoptogenic function. In contrast to cytochrome c, however, AIF acts in a caspase‐independent fashion. The molecular mechanisms via which AIF induces apoptosis are discussed.