Acyl-CoA dehydrogenases catalyze the a,ß-dehydrogenation of various acylCoA substrates. There are four known members of this flavoprotein family, which convert straight chain substrates. They can be subdivided according to the specificity for the chain 1ength of the latter. The best studied member of the this family is the homotetrameric medium-chain acyl-CoA dehydrogenase (MCAOH), which has a rather broad substrate chain length specificity centered around octanoyl-CoA (Cs-CoA). Oeficiency of this enzyme is the most common known genetic disorder in mitochondrial ß-oxidation in humans (I). About 90% of the reported cases involve a substitution of lysine to glutamic acid at position 304 of the mature enzyme (K304E-MCAOH), that is positioned at the interface between the subunits. The mutant protein shows a specific activity similar to that of wildtype MCAOH, but is only present at decreased levels in patient cells. This is due to impaired folding and tetramer assembly, and results in premature degradation of the enzyme. In addition to K304E-MCADH, new mutations have been found (2). Two of these are point mutations in the amino acid code of the mature protein resulting in the replacement of a cysteine at position 91 to a glycine or a tyrosine (Figure 1). ResuIts We have purified and partially characterized C9IG-MCAOH and C91YMCADH and compared these to hwt-MCADH. Both mutants are unstable. The activity of both mutants is decreased with the best substrates, compared to human, wild type MCADH (hwt-MCADH). Furthermore, the chain length/activity profile of C91G-MCADH is shifted towards long chain substrates. The instability is due to or accompanied by 10ss of FAD during purification.