Mitochondrial acetoacetyl-CoA thiolase (T2) deficiency is an inborn error of metabolism that affects the catabolism of isoleucine and ketone bodies. This disorder is characterized by intermittent ketoacidotic episodes. Recently, we diagnosed T2 deficiency in two patients (GK45 and GK47) by the absence of potassium ion-activated acetoacetyl-CoA thiolase activity, whereas these patients were previously misinterpreted as normal by a coupled assay with tiglyl-CoA as a substrate. This method has been widely used for the enzymatic diagnosis of the T2 deficiency in the United States and Europe. We hypothesized that some residual T2 activity showed normal results in the assay. To prove this hypothesis, we analyzed these two patients together with three typical T2-deficient patients (GK46, GK49, and GK50) at the DNA level. Expression analysis of mutant cDNAs clearly showed that GK45 and GK47 had "mild" mutations (A132G, D339-V340insD) that retained some residual T2 activity, at least one of two mutant alleles, whereas the other three patients had null mutations (c.52-53insC, G152A, H397D, and IVS8+1g>t) in either allele. These results raise the possibility that T2-deficient patients with mild mutations have been misinterpreted as normal by the coupled assay with tiglyl-CoA.