Newly identified forms of electron transfer flavoprotein (ETF) deficiency in two patients with glutaric aciduria type II (GA II) were described. GA II has been attributed to a defect of either ETF or ETF dehydrogenase, resulting in multiple acyl-CoA dehydrogenation deficiency. ETF is a mitochondrial flavoprotein consisting of an α-subunit, β-ETF, a α α-subunit, β-ETF. We used pulse-chase experiments to examine the biosynthesis of ETF in fibroblasts from two patients with GA II. Patient I was a boy with the neonatal onset form, but without congenital anomalies, who is living at age 2 y. A defect of β-ETF biosynthesis was noted in this patient. Patient 2 was a boy with the neonatal onset form with congenital anomalies who died on the 3rd postnatal day. He presented with a peculiar face and polycystic kidneys. In patient 2, both α-and β-ETF were synthesized, but both the subunits were rapidly degraded. The lability of ETF was considered to be the cause of GA II in this patient. These two cases appear to be new forms of ETF deficiency in GA II.