Arachidonoyl-CoA synthetase was solubilized from a particulate fraction of calf brain and human platelets using 1% Nonidet P-40 and 10 mM EDTA. Arachidonoyl-CoA synthetase from both preparations was separated from nonspecific (long chain) acyl-CoA synthetase (EC 6.2.1.3) by chromatography on hydroxylapatite. To further substantiate that the two acyl-CoA synthetases are distinct proteins, we solubilized enzyme from a mutant cell line lacking arachidonoyl-CoA synthetase and from the parent cell line from which it was derived. These preparations were chromatographed on hydroxylapatite, and the mutant showed an absence of the peak identified as arachidonoyl-CoA synthetase in the parent while retaining the peak of nonspecific acyl-CoA synthetase activity. We have also determined the levels of arachidonoyl and nonspecific acyl-CoA synthetase in 13 different human cells and tissues. Arachidonoyl-CoA synthetase is widely distributed and is present in significantly lower concentrations than nonspecific acyl-CoA synthetase only in adipose tissue and liver.