The determination of medium chain fatty acids in serum is a useful approach of the diagnosis of medium chain acyl-CoA dehydrogenase deficiency, an increasingly recognized cause of Reye-like syndrome in infants. A reliable and practical method requiring 0.5 ml of serum is presented by which results are obtained within 2.5 h. The preparation of the samples is done by solid phase extraction on reverse phase cartridges, the separation and quantitation by gas chromatography. Reference values for children (n = 24) and adults (n = 40) are given for octanoic, decanoic and dodecanoic acids.