The addition of 1-acyl-sn-glycero-3-phosphocholine (1-acyl-GPC) to peroxisomes decreased the production of acid-soluble radioactivity formed by β-oxidation of [1-14C]arachidonate due to substrate removal by esterification into the acceptor. This peroxisomal-associated acyl-CoA:1-acyl-GPC acyltransferase activity was due to microsomal contamination. The production of acid-soluble radioactivity from [1-14C]7,10,13,16–22:4, but not from [3-14C]7,10,13,16–22:4 was independent of 1-acyl-GPC, with and without microsomes. By comparing rates of peroxisomal β-oxidation with those for microsomal acylation, it was shown that the preferred metabolic fate of arachidonate, when added directly to incubations, or generated via β-oxidation, was esterification by microsomal 1-acyl-GPC acyltransferase, rather than continued peroxisomal β-oxidation.