Cellular energy metabolism is largely sustained by mitochondrial beta-oxidation of saturated and unsaturated fatty acids. To study the role of unsaturated fatty acids in cellular lipid and energy metabolism we generated a null allelic mouse, deficient in 3,2-trans-enoyl-CoA isomerase (ECI) (eci(-/-) mouse). ECI is the link in mitochondrial beta-oxidation of unsaturated and saturated fatty acids and essential for the complete degradation and for maximal energy yield. Mitochondrial beta-oxidation of unsaturated fatty acids is interrupted in eci(-/-)mice at the level of their respective 3-cis- or 3-trans-enoyl-CoA intermediates. Fasting eci(-/-) mice accumulate unsaturated fatty acyl groups in ester lipids and deposit large amounts of triglycerides in hepatocytes (steatosis). Gene expression studies revealed the induction of peroxisome proliferator-activated receptor activation in eci(-/-) mice together with peroxisomal beta- and microsomal omega-oxidation enzymes. Combined peroxisomal beta- and microsomal omega-oxidation of the 3-enoyl-CoA intermediates leads to a specific pattern of medium chain unsaturated dicarboxylic acids excreted in the urine in high concentration (dicarboxylic aciduria). The urinary dicarboxylate pattern is a reliable diagnostic marker of the ECI genetic defect. The eci(-/-) mouse might be a model of a yet undefined inborn mitochondrial beta-oxidation disorder lacking the enzyme link that channels the intermediates of unsaturated fatty acids into the beta-oxidation spiral of saturated fatty acids.