Medium-chain acyl-CoA dehydrogenase (MCADH) deficiency, an autosomal recessive inherited disorder, is the most common genetic disorder in mitochondrial beta-oxidation in humans. In addition to one prevalent disease-causing mutation (K304E), a series of rarer mutations has been reported, but none of these has yet been characterized in detail. We report here on the biochemical characterization of the purified recombinant mutant protein in which threonine is replaced by alanine at position 168 of the mature protein (T168A-MCADH). It is the first mutation to be found in patients that is located in the active site of the enzyme. Thr-168 is hydrogen-bonded to the flavin N(5) of the cofactor FAD. The thermostability of T168A-MCADH is markedly decreased compared with human wild-type MCADH (hwt-MCADH). Catalytic activity with ferricenium as acceptor is lowered by 80% and with the natural acceptor electron-transferring flavoprotein by over 90% compared with hwt-MCADH. In the mutant the extent of flavin semiquinone formed on reduction is approx. 50% that of hwt-MCADH. The pK reflected by the pH-dependence of Vmax is shifted from approx. 8.2 (hwt-MCADH) to approx. 7 (T168A-MCADH) and the rates of enzyme flavin reduction (stopped-flow measurements) are only approx. 1/10 those of the parent enzyme. These properties are discussed in the light of the possible mechanisms leading to disease in humans.