Mitochondrial trifunctional protein (TP) is an enzyme complex with three activities: enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, and 3-ketoacyl-CoA thiolase. Studies on defects in this enzyme in patients with TP deficiency suggest that there are two types of defect. Patients in group 1 have normal amount of cross-reacting material by immunoblot and lack only long-chain 3-hydroxyacyl-CoA dehydrogenase activity. Patients in group 2 have a trace amount of cross-reacting material, with all three activities being low. We identified three patients in group 2, and analysis was made at the cDNA level. In patient 2, there was a heterozygous 71-bp deletion at position 110-180 in the alpha-subunit. In patients 1 and 3, there was an abnormal beta-subunit; patient 1 had an A-788-to-G substitution, and patient 3 had G-182-to-A and G-740-to-A substitutions in each of separate alleles. This is the first demonstration of disease-causing mutations in the beta-subunit. cDNA-expression experiments in patients' fibroblasts, using a vaccinia virus system, and gel filtration analysis, using patients' fibroblasts, revealed that the existence of both normal alpha- and beta-subunits, and possibly their association, are important for stabilizing TP and that A-788-to-G substitution on the beta-subunit in patient 1 seems to interfere with the association, the result being a rapid decomposition of TP.