The peroxisomal beta-oxidation system consists of four steps catalysed by three enzymes: acyl-CoA oxidase, 3-hydroxyacyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase (multifunctional enzyme) and thiolase. In humans, thiolase activity is encoded by one gene, whereas in rodents, three enzymes encoded by three distinct genes (i.e. thiolase A, thiolase B and SCP2/thiolase) catalyse the thiolase activity. So far, acyl-CoA oxidase- and multifunctional enzyme-deficient patients have been identified and knock-out mice for these genes have been produced. Conversely, no isolated thiolase-deficient patient has been found, and no thiolase (A or B)-deficient mice have been generated. Hence, to better understand the cause of isolated human thiolase deficiency, we disrupted the catalytic site of the mouse thiolase B by homologous recombination in order to analyse the phenotype of these thiolase B-deficient mice. Mice, made homozygous for the mutation, lack expression of thiolase B mRNA and are viable, fertile and healthy at birth. They exhibit no detectable phenotype defects and no compensation, rather a slight decrease in other peroxisomal thiolase (thiolase A and SCPx) mRNAs, was found.