Using neuronal nuclei (N1) and microsomes (P3) isolated from cerebral cortices of 15-day-old rabbits, the activity of lysophosphatidylcholine acyltransferase (acyl CoA: 1-acyl-sn-glycerol-3-phosphorylcholine acyltransferase) was studied using palmitoyl-, oleoyl- and arachidonoyl-CoA and a pool of lysophosphatidylcholine labelled with [3H]palmitate, [3H]stearate or [3H]oleate. Generally, in the acylation of the three radioactive lysophosphatidylcholines with arachidonoyl-CoA, the N1-specific acylation activities were two to seven times those of P3. For oleoyl-coA smaller N1 : P3 specific activity ratios were found, differing significantly from unity for only the 1-palmitoyl and 1-stearoyl lysophosphatidylcholines. The N1 : P3 ratios for the two unsaturated acyl-CoA thioesters were usually found to increase as the lysophosphatidylcholine concentration was lowered from 100 to 25 microM. Thus, nuclear acylation rates, particularly with arachidonoyl-CoA, were less affected by lowering the acceptor concentration than were microsomal activities. At the high lysophosphatidylcholine concentration (100 microM), arachidonoyl-CoA was a superior substrate to oleoyl-CoA in the nuclear acylations of the 1-palmitoyl or 1-stearoyl acceptors. Such a preference was never seen for the microsomal fraction. Using oleoyl- and arachidonoyl-CoA, the nuclear enzymes also showed a greater preference for the 1-palmitoyl homologue over the 1-oleoyl homologue than did the microsomal enzymes. These results support the existence of neuronal nuclear lysophosphatidylcholine acyltransferases with different substrate preferences than shown by the microsomal fraction.