In most biological materials, free L-carnitine is present together with short-chain and long-chain carnitine esters. These are differentiated mainly according to their solubility in aqueous solvents. A standardized extraction procedure is therefore essential for reproducible estimations of carnitine content. Assays of L-carnitine are based on the reaction of L-carnitine with acetyl CoA with formation of acetyl L-carnitine and free CoASH, catalysed by carnitine acetyl transferase (EC 2.3.1.7). The two main principles employed to monitor this reaction are a) measurement of the incorporation of radio-labelled acetyl groups derived from acetyl CoA into acetyl carnitine, and b) photometric determination of free CoASH formed in the reaction, using thiol-group colour reagents or an enzymatic reaction. To avoid the background due to thiol-compounds in the sample, we suggest the introduction of an oxidation step with hydrogen peroxide, which is then removed with catalase. Using this method, we have established reference ranges for total acid-soluble L-carnitine and free L-carnitine in serum (men: 44.2-79.3, and 34.8-69.5, women: 28.1-66.4 and 19.3-53.9 mumol/l, resp.), skeletal muscle (adults: 21.0-23.1, and 19.5-35.1, children: 16.1-39.0 and 12.1-25.5 mumol/g non-collagen protein, resp.), and urine. The concentration of long-chain acyl L-carnitine in serum is 2.0-4.0 mumol/l. Carnitine levels in serum, tissues and urine are age-dependent with lower levels in newborn children. The ratio of short-chain acyl carnitine to free carnitine is mainly a reflection of hepatic acetyl CoA production.(ABSTRACT TRUNCATED AT 250 WORDS)