Fish-odour syndrome is a highly unpleasant disorder of hepatic trimethylamine (TMA) metabolism characterized by a body odour reminiscent of rotting fish, due to excessive excretion of the malodorous free amine. Although fish-odour syndrome may exhibit as sequelae with other conditions (e.g. liver dysfunction), many patients exhibit an inherited, more persistent form of the disease. Ordinarily, dietary-derived TMA is oxidized to the non-odorousN-oxide by hepatic flavin-containing monooxygenase 3 (FMO3). Our previous demonstration that a mutation, P153L (C to T), in theFMO3gene segregated with the disorder and inactivated the enzyme confirmed that defects inFMO3underlie the inherited form of fish-odour syndrome. We have investigated the genetic basis of the disorder in two further affected pedigrees and report that the three propositi are all compound heterozygotes for causative mutations ofFMO3. Two of these individuals possess the P153L (C to T) mutation and a novel mutation, N61S (A to G). The third is heterozygous for novel, M434I (G to A), and previously reported, R492W (C to T), mutations. Functional characterization of the S61, I434 and W492 variants, via baculovirus-mediated expression in insect cells, confirmed that all three mutations either abolished, or severely attenuated, the capacity of the enzyme to catalyse TMAN-oxidation. Although I434 and W492 were also incapable of catalysing theS-oxidation of methimazole, S61 was fully active with this sulphur-containing substrate. Since an asparagine is conserved at the equivalent position to N61 of FMO3 in mammalian, yeast andCaenorhabditis elegansFMOs, the characterization of the naturally occurring N61S (A to G) mutation may have identified this asparagine as playing a critical role specifically in FMO-catalysedN-oxidation.