Apoptosis‐inducing factor (AIF) is a mitochondrial intermembrane flavoprotein that is translocated to the nucleus in response to proapoptotic stimuli, where it induces nuclear apoptosis. Here we show that AIF is synthesized as an ∼67‐kDa preprotein with an N‐terminal extension and imported into mitochondria, where it is processed to the ∼62‐kDa mature form. Topology analysis revealed that mature AIF is a type‐I inner membrane protein with the N‐terminus exposed to the matrix and the C‐terminal portion to the intermembrane space. Upon induction of apoptosis, processing of mature AIF to an ∼57‐kDa form occurred caspase‐independently in the intermembrane space, releasing the processed form into the cytoplasm. Bcl‐2 or Bcl‐XL inhibited both these events. These findings indicate that AIF release from mitochondria occurs by a two‐step process: detachment from the inner membrane by apoptosis‐induced processing in the intermembrane space and translocation into the cytoplasm. The results also suggest the presence of a unique protease that is regulated by proapoptotic stimuli in caspase‐independent cell death.