We have recently noted that cells treated with fish oil and n-3-fatty acids show slower DNA replication rates than cells treated with a control emulsion or corn oil only. However, it is not clearly understood how such an effect is induced. Fish oil and its metabolites are known to have several modulating effects on signal transduction pathways. Alternatively, they may influence DNA replication by interacting directly with nuclear components. To investigate this problem in greater detail, we have studied the kinetics of DNA synthesis in a cell-free system derived from HeLa cells. Nuclei and cytosolic extract were isolated from cells synchronized in early S phase after treatment with control emulsion, corn oil, or fish oil, respectively. The nuclei were reconstituted with cytosolic extract and a reaction mixture containing bromodeoxyuridine (BrdU) triphosphate to label newly synthesized DNA. The rate of DNA synthesis was measured by bivariate DNA/BrdU analysis and flow cytometry. We show that fish oil-treated cytosol inhibits the elongation of newly synthesized DNA by ~80% in control nuclei. However, nuclei treated with fish oil escape this inhibitory effect. We also show that addition of nuclear extract from fish oil-treated cells reverses the inhibitory effect seen in the reconstitution system of control nuclei and fish oil-treated cytosol. These results indicate that polyunsaturated fatty acids can modulate DNA synthesis through cytosolic as well as soluble nuclear factors.