Tris and HEPES were systematically compared as buffers for the enzymatic assay of L-carnitine. The deproteinization methods preceding the assay were also compared. The following conclusions were drawn. 1. Both Tris and HEPES act on the catalytic site of the enzyme, acetylCoA: carnitine O-acetyltransferase (EC 2.3.1.7), which is used for the conversion of L-carnitine to acetylcarnitine. HEPES is a competitive inhibitor, and no acetylated product of HEPES is formed. In the presence of Tris a limited amount of acetylTris is formed, and an appropriate blank corrects for this effect. 2. The incubation time of the assay is strongly influenced by the preceding deproteinization method. The enzyme is influenced by inorganic salt, which acts as a competitive inhibitor. 3. If Tris is used in place of HEPES in end-point assays, optimal conditions and shorter assay times are achieved with less enzyme and less acetylCoA, provided more elaborate deproteinization methods are used. 4. The HEPES system is more costly, but preferable for the determination of both total and free L-carnitine in combination with a matched deproteinization method.