Mitochondrial preparations from riboflavin-deficient rats are known to exhibit reduced capacity for the oxidation of fatty acids. During a search for a suitable fatty acid probe for the in vivo exploration of this phenomenon, it became apparent that a dicarboxylic acid, adipic acid, was more promising than a monocarboxylic acid such as octanoic acid. Rats made riboflavin deficient, and controls at various stages of development, were dosed intragastrically with [1,6-14C]adipic acid, and 14CO2 production was measured for 3 h. All animals were then given flavin mononucleotide intragastrically to replete their tissues, and the [14C]adipic acid test was repeated at intervals thereafter. In all groups, repletion produced a highly significant increase in the whole-body adipic acid oxidation index, and reversed the low rates of oxidation which had been observed in nonrepleted, deficient animals. This approach may therefore permit the development of a new functional test for fatty acid oxidation in human riboflavin deficiency, and for other conditions in which fatty acid oxidation pathways are impaired, by the use of nonradioactive [13C]adipic acid.