As part of a long-term study of sphingolipid metabolism in brain, we have purified and partially characterized a long-chain acyl-CoA synthetase from microsomes of developing rat brain and compared it with the hepatic microsomal enzyme from the same animals. Both enzymes were solubilized from microsomes by treatment with Triton X-100 and then chromatographed successively on Blue-Sepharose and DEAE-Sepharose. Blue-Sepharose chromatography yielded a single peak with acyl-CoA synthetase activity, whereas DEAE-Sepharose chromatography of both brain and liver preparations yielded two peaks. Elution patterns of lignoceroyl-CoA synthetase and palmitoyl-CoA synthetase activities were identical throughout these steps and were similar in brain and liver. Gel filtration of each DEAE-Sepharose fraction on Sephadex G-200 also yielded two peaks of activity. The more rapidly eluted material contained much more lignoceroyl-CoA synthetase activity, while the activity for palmitoyl-CoA synthetase was higher in slower eluting peaks. In all preparations the ratio of lignoceroyl-CoA synthetase activity to palmitoyl-CoA synthetase activity was much higher in brain than in liver. These results suggest that although the brain acyl-CoA synthetase is chromatographically similar to the liver enzyme, there are differences in substrate specificity.