Mitochondrial fatty acid β-oxidation is a crucial source of energy for many mammalian tissues. The trifunctional protein (TFP) is a heteroctamer of 4α and 4β subunits which catalyzes three steps in this pathway for long chain fatty acids. The α subunit contains the long chain enoyl-CoA hydratase and the long chain 3-hydroxy-acyl-CoA dehydrogenase (LCHAD) activities. The β subunit catalyzes the long chain 3-keto-thiolase cleavage step. Deficiency in the TFP in humans causes Sudden Infant Death Syndrome, acute fatty liver (Reye-like syndrome), and cardiomyopathy. We previously cloned the mouse and human cDNAs and genes encoding the α subunit. The mouse β subunit cDNA is 2 kb in length and has 16 exons. We characterized the 15 kb mouse and human thiolase genes, each of which contains 16 exons. We found that the 5' end of the human β subunit gene is located 450 bp upstream of the α subunit gene. The two genes are arranged in opposite orientations, suggesting that they may be coordinately regulated. We also screened patients for mutations in the β subunit gene using single stranded conformation variance (SSCV). The mutations and polymorphisms were verified by automatic sequencing. One patient with peripheral myopathy is heterozygous for two mutations in the β subunit, G182A in exon 4, R128H, and A740G in exon 9, R214A. We also found two polymorphisms. The first, T825C in exon 10, does not alter a Val codon. The second is an ACT deletion that removes a Thr from position three of transit peptide. We found that 19% of the population has 1 Thr in the transit peptide of TFP and that 81% has 2 Thr residues. Mitochondrial uptake studies showed that the peptide with 2 Thr residues is incorporated more efficiently. Also, one patient deficient in TFP had no other mutations in the α or β subunit genes and was homozygous for 1 Thr. This suggests that the detrimental form of the allele is the one with only one Thr, and that with the second Thr, the precursor protein's rate of mitochondrial uptake is decreased significantly enough to induce the disease phenotype. Thus, we have found two patients with long chain 3-keto-acyl-CoA thiolase deficiency. The first is a compound heterozygote with a substituted amino acid in exon 4 and one in exon 9. The second is a homozygote with a deletion in each copy of his β subunit transit peptide.