Mitochondrial trifunctional protein is a hetero-octamer of 4 α- and 4β-subunits that catalyze the final 3 steps in the β-oxidation spiral of long chain fatty acids. The α-subunit contains the activities of the long chain 2-enoyl-CoA hydratase and the long chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD); while the activity of the long chain 3-ketoacyl-CoA thiolase resides in the β-subunit. We used single strand conformation variance analysis and nucleotide sequencing to identify the genetic defects in 18 patients with TFP deficiency or isolated LCHAD deficiency proven by enzymatic assays. Three patients presented with neonatal dilated cardiomyopathy in the first month of life. All these patients had TFP deficiency secondary to missplice or missense mutations in exons 3, 7, or 19. Thirteen patients with an isolated LCHAD deficiency presented predominantly with hepatic dysfunction and Reye-like syndrome at mean age of 5 months. Eight of these patients were homozygous for a common exon 15 mutation (G1528C, E474Q). The remaining 5 patients were heterozygotes with one allele carrying the G1528C mutation, while the mutations in the other allele were in exons 5, 6, 12, 16, or 18 causing stop codons and premature termination. Allele specific oligonucleotide hybridization studies revealed undetectable levels of mRNA carrying the stop codon mutations. The maternal pregnancy history revealed development of acute fatty liver of pregnancy (AFLP) and/or HELLP(hemolysis, elevated liver enzymes, and low platelets) in eleven heterozygote mothers who carried fetuses diagnosed later to have isolated LCHAD deficiency. Two unrelated patients presented with unique chronic phenotype at several years of age characterized by slowly progressive myopathy and neuropathy without significant cardiac or hepatic involvement. Both patients had TFP deficiency secondary to missense mutations in exon 9 which codes for a linker domain between the hydratase and the LCHAD domains in the matureα-subunit. These results document the existence of novel genotype-phenotype correlations in pediatric TFP deficiency.