The trafficking of acyl-CoAs within cells is poorly understood. In order to determine whether newly synthesized acyl-CoAs are equally available for the synthesis of all glycerolipids and cholesterol esters, we incubated human fibroblasts with [¹⁴C]oleate, [³H]arachidonate or [³H]glycerol in the presence or absence of triacsin C, a fungal metabolite that is a competitive inhibitor of acyl-CoA synthetase. Triacsin C inhibited de novo synthesis from glycerol of triacylglycerol, diacylglycerol and cholesterol esters by more than 93%, and the synthesis of phospholipid by 83%. However, the incorporation of oleate or arachidonate into phospholipids appeared to be relatively unimpaired when triacsin was present. Diacylglycerol acyltransferase and lysophosphatidylcholine acyltransferase had similar dependences on palmitoyl-CoA in both liver and fibroblasts; thus it did not appear that acyl-CoAs, when present at low concentrations, would be preferentially used to acylate lysophospholipids. We interpret these data to mean that, when fatty acid is not limiting, triacsin blocks the acylation of glycerol 3-phosphate and diacylglycerol, but not the reacylation of lysophospholipids. Two explanations are possible: (1) different acyl-CoA synthetases exist that vary in their sensitivity to triacsin; (2) an independent mechanism channels acyl-CoA towards phospholipid synthesis when little acyl-CoA is available. In either case, the acyl-CoAs available to acylate cholesterol, glycerol 3-phosphate, lysophosphatidic acid and diacylglycerol and those acyl-CoAs that are used by lysophospholipid acyltransferases and by ceramide N-acyltransferase must reside in two non-mixing acyl-CoA pools or, when acyl-CoAs are limiting, they must be selectively channelled towards specific acyltransferase reactions.