A new acylcarnitine was observed in the plasma and urine of a patient with isolated 3-methylcrotonyl-CoA carboxylase deficiency. Analysis by tandem mass spectrometry of the methyl ester and butyl ester and their fragment ion spectra identified it as a 3-hydroxy-C5-acylcarnitine. Fibroblasts from a second patient were incubated with deuterium-labelled leucine. Incorporation of label in the new acylcarnitine identified its origin from leucine, and thus confirmed the structure as 3-hydroxyisovalerylcarnitine. The presence of elevated amounts of this metabolite, plus a small amount of 3-methylcrotonylcarnitine in plasma, was diagnostic for isolated 3-methylcrotonyl-CoA carboxylase deficiency. Other conditions in which a hydroxy-C5-acylcarnitine was present were readily differentiated by the abnormal elevation of other acylcarnitines.