Carnitine palmitoyltransferase (CPT) has been purified, and some of its properties are described. The purified preparations had specific activities of 10–38 μmoles/min/mg protein at 35°. The apparent molecular weight of CPT was approximately 75 000, or a multiple thereof. The K′m value for palmitoyl-CoA was 1.8 × 10−5 M, while the K′s values for (–)-carnitine, CoA–SH, and (–)-palmitoylcarnitine were estimated to be 4.5 × 10−4 M, 5.5 × 10−6 M, and 1.4 × 10−4 M, respectively. The enzyme was subject to inhibition by all substrates and products. CPT had a relatively broad fatty acyl chain-length specificity, with highest Vmax values obtained for the transfer of myristoyl and palmitoyl groups. K′m values were lowest with long-chain fatty acylcarnitinè derivatives (1.9 × 10−4 M for (–)-stearoylcarnitine versus 1.2 × 10−3 M for (–)-decanoylcarnitine).Evidence is presented demonstrating the existence of two additional protein fractions possessing carnitine acyltransferase activities with different fatty acyl chain-length specificities. One, which displayed highest relative activity towards the transfer of octanoyl groups, is tentatively designated carnitine octanoyltransferase. The other, which displayed a high specificity toward the transfer of palmitoyl and stearoyl groups from acylcarnitine when preincubated with CoA–SH, is tentatively designated carnitine palmitoyltransferase II.