4-Heptanone is a common volatile constituent of human urine and is of unknown origin. We hypothesised that it arises from in vivo β-oxidation of 2-ethylhexanoic acid (EHA) from plasticisers, similar to formation of 3-heptanone from valproic acid. We investigated urine from individuals with normal and increased plasticiser exposure. Using GC/MS, solvent-extracted organic acids were analysed as trimethylsilyl (TMS) derivatives and heptanone with headspace solid-phase microextraction. We identified 3-oxo-2-ethylhexanoic acid, the β-oxidation product of EHA, as an enol in all samples. This is the first report of its TMS mass spectrum. We also found 2-ethyl-1,6-hexanedioic acid and 5-hydroxyEHA, ω- and ω-1-oxidation products of EHA, respectively, and 2-ethylhexanoylglucuronide, but only in trace amounts in some plasticiser samples. These compounds have not been reported in human urine, nor has the TMS mass spectrum of 5-hydroxyEHA. The median concentrations of 3-oxoethylhexanoic acid and total 4-heptanone of seven plasticiser samples were around 30–175-fold higher than normal samples. 4-Heptanone was barely detectable and 3-oxoethylhexanoic acid was not increased in an eighth plasticiser sample, from a baby with deficiency of 2-methylbranched-chain acyl-CoA dehydrogenase. β-Oxidation is a major catabolic pathway of EHA in man, and might be involved in the metabolism of other branched-chain drugs and environmental pollutants.