We previously reported that unsaturated long-chain fatty acids were potent DNA polymerase inhibitors (Y. Mizushina et al., J. Biol. Chem. 274 (1999) 25599-25607). In those experiments, the question remained of whether metastable oil droplets (liposomal vesicles) of the unsaturated long-chain fatty acids can non-specifically inhibit the polymerase activity. We report here that only the soluble fatty acid monomers of linoleic acid or nervonic acid could affect the activities of mammalian DNA polymerases, and the metastable oil droplets could not. When we consider the facts that nuclear membranes are a kind of liposomal vesicles, that free fatty acids occur only at the moment the lipids are digested, and that the DNA polymerization possibly occurs on the nuclear membranes, the data shown here are suggestive regarding the mechanism of regulation of DNA polymerization in vivo.