The substrate specificity of fatty acid elongase was studied using an oil body fraction from developing seeds of Brassica napus. ATP was essential for high rates of elongase activity, but there was no apparent requirement for oleoyl-CoA, oleic acid (18:1) or CoA. Furthermore, 14C from 18:1-CoA was incorporated into eicosenoic (20:1) and erucic (22:1) acids at a much slower rate than 14C from malonyl-CoA. Incubation of [14C]18:1-CoA with the oil body fraction resulted in a rapid loss of [14C]18:1-CoA into several lipid fractions whether in the absence or presence of ATP, but the loss of 18:1-CoA had a comparatively small effect on the overall rate of elongation. Acyl-CoAs were derivatized to their respective acylbutylamide and analyzed by gas chromatography-mass spectrometry. This analysis of acyl-CoAs demonstrated that there was no detectable 20:1-CoA or 22:1-CoA at 0 min incubation, while newly synthesized 20:1-CoA and 22:1-CoA were present at 10 min. Analysis of the %14C of the substrates and products of the elongation reaction revealed that the endogenous pool of 18:1-CoA is quite small in elongase preparations. In addition, [14C]18:1-CoA added to the incubation, although incorporated into lipids, was not significantly diluted by turnover or new synthesis. In contrast, the %14C of the 20:1-CoA was two- to threefold less than that of the 18:1-CoA. Taken together, these results indicate that the [14C]18:1 from the [14C]18:1-CoA was diluted in an intermediate 18:1 pool and that the 18:1-CoA was not the major donor of the acyl group to the elongase reaction.