The activity of medium-chain acyl-CoA dehydrogenase (MCAD) with octanoyl-CoA as a substrate was measured in human lymphocytes by a gas chromatographic technique. Phenazine methosulfate was used as the primary electron acceptor. After the addition of crotonase and subsequent hydrolysis, the reaction product 3-hydroxyoctanoic acid was quantitated by capillary gas-liquid chromatography of the trimethylsilyl derivatives. Control subjects had MCAD activities of 3.46 +/- 0.18 nmol/mg protein/min (n = 15). Five patients were investigated while receiving no therapy at all; MCAD activity ranged from 0.08 to 0.23 in four of them and was 0.65 in the fifth one. Subsequent to the long-term administration of 50-150 mg/d of riboflavin to MCAD-deficient patients (n = 11), these activities increased to an average of 0.41 in 10 patients and 2.22 in one. The activities in 15 obligate heterozygotes were 1.91 +/- 0.41 nmol/mg protein/min, thus enabling a clear distinction from controls. Neither heterozygotes nor a control responded to riboflavin. The method was also applicable to postmortem liver tissue. One patient, who had died suddenly and unexpectedly at the age of 19 mo, was correctly diagnosed as MCAD-deficient, whereas five additional children who died of the sudden infant death syndrome showed normal activities.