The deduced full-length ACAD11 protein contains an N-terminal aminoglycoside phosphotransferase domain, followed by a mitochondria localization signal and ACAD N-terminal, middle, and C-terminal domains. It also has a glycosylation site and a C-terminal peroxisome-targeting signal. The ACAD domain contains a catalytic aspartate rather than the catalytic glutamate found in most other ACADs. Real-time RT-PCR detected variable ACAD11 expression in all tissues examined, with highest expression in adult brain, followed by heart, liver, spinal cord, and kidney. Western blot analysis of fractionated human cerebellum and kidney revealed that ACAD11 associated with mitochondrial membranes, but not mitochondrial matrix. Immunohistochemical analysis showed that ACAD11 colocalized with a mitochondrial marker in human neuroblastoma cells.