The concentrations of 2-methylacetoacetate, 2-methyl-3-hydroxybutyrate and tiglylglycine were determined by gas chromatography-mass spectrometry in urine collected before and for 8 h after loading with 100 mg of isoleucine per kg of body weight. The sum of 2-methylacetoacetate and 2-butanone, a decarboxylation product, was determined as the 2-butanone dinitrophenylhydrazone derivative. Substantial increases in each compound were encountered in a patient with a documented defect of 2-methylacetoacetyl-CoA thiolase. Increased quantities of 2-methyl-3-hydroxybutyrate and tiglylglycine were also found in four children with clinical symptoms similar to those associated with 2-methylacetoacetyl-CoA thiolase deficiency but in whom the activity of the enzyme was found to be normal. The concentration of 2-methylacetoacetate plus 2-butanone in the urine increased after an isoleucine load only in the patient with 2-methylacetoacetyl-CoA thiolase deficiency.