The level of carnitine in serum was examined in a control group and in different patient groups. A radiometric assay of carnitine with carnitine acetyltransferase and radioactively labeled acetyl-CoA was used. By the addition of dithionitrobenzoic acid (DTNB) to the incubation mixture to trap the CoASH released, the formation of radioactive acetylcarnitine was proportional to the carnitine concentration over a wide range. Carnitine was extracted from plasma after ethanol addition and the recovery of [Me-³H] carnitine added to serum was 102%. The serum level of carnitine was reduced in patients with myotonia congenita, Crohn's disease in some patients with malabsorption and in cases of anorexia nervosa. During hemodialysis the carnitine concentration was reduced to 25% of the value occurring before dialysis. Normal levels of carnitine were found in ulcerative colitis patients, most patients with malabsorption, in liver cirrhosis, in right-sided heart failure with liver stasis, in juvenile and adult onset diabetes, and in cardiomyopathies. Two patients with progressive cardiac failure with icterus and oliguria showed an eight-fold increase in serum carnitine levels.