In a previous paper, we showed inhibition of rat liver L-threonine dehydrogenase by a preparation obtained by dialysis and concentration from rat liver mitochondria stored at -20 degrees C for 7-10 days (Pagani, R., Leoncini, R., Guerranti, R. and Marinello, E. (1990) It. J. Biochem. 39, 106-114). The chemical composition of the fraction containing the unknown 'inhibitor' has now been studied and identified as D-3-hydroxybutyrate (D3HB).