Long-chain acyl-CoA synthetase (EC 6.2.1.3), an enzyme(s) that activates fatty acids prior to incorporation into phospholipids and other substances, has been detected in highly purified myelin from rat brain stem. The high levels relative to microsomes (11% and 15% for oleate and arachidonate, respectively) tended to preclude contamination by the latter membrane as the source of activity. Additional evidence came from sequential purification and mixing experiments. Km values were not appreciably different for the two substrates with the two membranes, but Vmax values were approximately 2-4-fold greater for arachidonate in both membranes. Triton X-100 increased activity somewhat in myelin but not in microsomes; with arachidonate as substrate it reduced activity in the latter. Heat inactivation studies and pH profiles suggested the presence of two different enzymes, as previously shown for other tissues.