A long-chain fatty acyl-CoA synthetase that catalyzes the activation of long-chain fatty acids as thioesters of CoA, was described in rat liver nuclei. This is the first step for further metabolization of fatty acids in the cell. Up to now, it has been shown that long-chain fatty acyl-CoA synthetase is located in the endoplasmic reticulum, in plasma membrane, in mitochondria and in peroxisomes. The nuclear long-chain fatty acyl-CoA synthetase was assayed using palmitic (16:0), linoleic (18:2n-6) and 8,11,14-eicosatrienoic (20:3n-6) acids as substrates and was stimulated linearly with nuclear protein concentration and with incubation time The higher enzymatic activity was observed with 18:2n-6 and 20:3n-6 acids as substrates. The synthesis of palmitoyl-CoA, linoleyl-CoA and 8,11,14-eicosatrienoyl-CoA followed normal Michaelis-Menten kinetics with respect to the corresponding substrate concentrations. The acyl-CoA synthetase seems to be saturated at a substrate concentration of 12.8 microM for all the acids tested. The apparent Km values decreased in the following order 20:3n-6 > 18:2n-6 > 16:0. The lowest apparent Km for palmitic acid indicates a preference for acylation of this acid in the cell nucleus.