We report clinical and biological investigations in two patients (twin brothers) with 2-methylacetoacetyl-CoA thiolase deficiency. Main clinical features included important staturo-ponderal delay, frequent infectious rhinopharyngitis episodes and an acute metabolic acidosis at the age of 4 years, this metabolic decompensation being adequately halted by bicarbonate supplementation. Since that age, patients developed rather favorably, however, with persistence of the staturo-ponderal delay. Organicaciduria typical of 2-methylacetoacetyl-CoA thiolase deficiency was recorded consisting of excessive excretion of tiglylglycine, 2-methyl-3-hydroxybutyrate, 3-hydroxyisovalerate, 2-methylglutaconate, adipate and 2-methylacetoacetate. Blood carnitine levels were altered in patients with increased total and esterified carnitine concentrations and enhanced acyl/free carnitine ratios. Determination of acylcarnitine profiles showed that patients excreted excessive amounts of several acylcarnitines in urine including propionyl, butyryl, isobutyryl, isovaleryl, 2-methylbutyryl and tiglyl-carnitine, the latter acylcarnitine being prominent with, in one of the patients, occurrence of a previously undescribed isomer of this carnitine ester, possibly 2-ethylacrylyl-carnitine. Excretion of these acylcarnitines in urine was increased in response to L-carnitine although, as a whole, this therapy resulted in a less important stimulation of esterified carnitine removal in urine from patients than in the case of supplemented controls. Biochemical investigations on cultured skin fibroblasts confirmed 2-methylacetoacetyl-CoA thiolase deficiency. Through the present report on this rare disease in two siblings, we would like to underline that acylcarnitines can be used in the diagnosis of 2-methylacetoacetyl-CoA thiolase deficiency, a view supported by acylcarnitine profiles further determined in another patient with proven oxothiolase deficiency, adding this pathology to the list of beta-oxidation disorders that may be screened successfully through determination of acylcarnitine profiles in body fluids.