Trifunctional protein (TFP) plays a significant role in the mitochondrial beta-oxidation of long-chain fatty acids. Its deficiency impairs the energy generating function of this pathway and causes hypoketotic hypoglycemia once hepatic glycogen stores are depleted. A Reye-like syndrome, cardiomyopathy, and sudden death may follow. The diagnosis is based on demonstration of significantly decreased enzyme activity of at least two of the three involved enzymes in fibroblasts. The possibility of prospective diagnosis of TFP deficiency by newborn screening using tandem mass spectrometry (MS/MS) has not been evaluated. We report the postmortem diagnosis of a male newborn, who suffered sudden death at 2 wk of age, and his younger sister, who died of cardiomyopathy complicated by acute heart failure at the age of 6 mo, after she had acquired a common viral infection. Blood spots from the original newborn screening cards were the only remaining material from the patients. Analysis by MS/MS revealed acylcarnitine profiles consistent with either TFP or long-chain 3-hydroxyacyl coenzyme A dehydrogenase (LCHAD) deficiency. To prove the diagnosis, the alpha- and beta-subunit genes coding for TFP were examined. The patients were compound heterozygous for a 4-bp-deletion and an a-->g missense mutation, both in the same exon 3 donor consensus splice site. This is the first report of the diagnosis of TFP deficiency using blood spots obtained for newborn screening and suggests that TFP deficiency may be detectable by prospective newborn screening using MS/MS.