C6-C10-dicarboxylic and C6-C10-ω-1-hydroxy monocarboxylic acids were measured in postmitochondrial (10,000 g) fractions of rat liver after incubation with hexanoic, octanoic, and decanoic acids. In livers both from fed and starved rats, the proportion of decanoic acid converted to sebacic acid was high (approximately 25%) with only minor accumulation of the intermediate 10-hydroxy decanoic acid (1–2%). The conversion of octanoic and hexanoic acids to suberic and adipic acids, respectively, was low (i.e.,171 and 3.1 μmole/liter, respectively, whereas they are high for octanoic and hexanoic acids (8211 and 8822 μmole/liter, respectively). In two different humans, the corresponding Km values for dodecanoic, decanoic, octanoic, and hexanoic acids are 3.6–186, 522–247, 4861–3892, and 6825–10400 μmole/liter, respectively. Based on these results, it is argued that adipic and suberic acids found in urine from rats and humans with acyl-CoA dehydrogenation deficiencies are not biosynthesized by direct ω-oxidation of hexanoic and octanoic acids, but most probably by means of β-oxidation of sebacic and dodecanedioic acids, produced by direct ω-oxidation.The affinities of the ω-1-hydroxylation systems for the four monocarboxylic acids is similar in rats and humans. The apparent Km values for dodecanoic acid in rats and in the two human livers are low,i.e.,139 and 92–131 μmole/liter, respectively. The Km values for decanoic, octanoic, and hexanoic acids are all high in both rats and humans,i.e.,in the range from 638 μmole/liter (for decanoic in one of the human livers) to more than 20,000 μmole/liter (for hexanoic acid in both species). It is argued that 5-hydroxy hexanoic, 7-hydroxy octanoic, and 9-hydroxy decanoic acids found in some patients with acyl-CoA dehydrogenation deficiencies are produced by β-oxidation of 11-hydroxy dodecanoic acid, which may be produced directly by ω-1-hydroxylationof dodecanoic acid.