The complete AIF cDNA comprising the ammo‐terminal mitochondrial localization sequence (MLS) and the oxidoreductase domain has been fused in its carboxyl terminus to enhanced green fluorescent protein (GFP), thereby engineering an AIF‐GFP fusion protein that is selectively targeted to the mitochondrial intermembrane space. Upon induction of apoptosis, the AIF‐GFP protein translocates together with cyto¬chrome c (Cyt‐c) to the extramitochondrial compart¬ment. Microinjection of recombinant AIF leads to the release of AIF‐GFP and Cyt‐c‐GFP, indicating that ectopic AIF can favor permeabilization of the outer mitochondrial membrane. These mitochondrial effects of AIF are caspase independent, whereas the Cyt‐c‐microinjection induced translocation of AIF‐GFP and Cyt‐c‐GFP is suppressed by the pan‐caspase inhibitor Z‐VAD.fmk. Upon prolonged culture, transfection‐enforced overexpression of AIF results in spontaneous translocation of AIF‐GFP from mitochondria, nuclear chromatin condensation, and cell death. These effects are caspase independent and do not rely on the oxidoreductase function of AIF. Spontaneous AIF‐GFP translocation and subsequent nuclear apoptosis can be retarded by overexpression of a Bcl‐2 protein selec¬tively targeted to mitochondria, but not by a Bcl‐2 protein targeted to the endoplasmic reticulum. Over¬expression of a mutant AIF protein in which the MLS has been deleted (AIF Δ 1–100) results in the primary cytosolic accumulation of AIF. AIF Δ 1–100‐induced cell death is suppressed by neither Z‐VAD.fmk or by Bcl‐2. Thus, extramitochondrially targeted AIF is a dominant cell death inducer.—Loeffler, M., Daugas, E., Susin, S. A., Zamzami, N., Metivier, D., Nieminen, A.‐L., Brothers, G., Penninger, J. M., Kroemer, G. Dominant cell death induction by extramitochondrially targeted apoptosis‐inducing factor. FASEB J. 15, 758‐767 (2001)