Fish oils have been noted to reduce the growth and proliferation of certain tumor cells. These effects are usually attributed to the content of polyunsaturated fatty acids of the 3 family, which are thought to modulate cellular signaling pathways. We investigated the influence of fish oil on the cell cycle kinetics of cultured Chinese Hamster Ovary cells. Exponentially growing cells were labeled with bromodeoxyuridine (BrdUrd) and analyzed by flow cytometry after a 5-day treatment with exogenous fat. Bivariate BrdUrd/DNA analysis indicated a slower progression through S-phase and thus, a longer S phase duration time in fish oil treated cells but not in corn oil treated or control cells. We hypothesize that fish oil treatment might interfere with the spatial/temporal organization of initiation of replication origins. Therefore, we mapped the well-characterized replication origin ori-ß downstream of the dihydrofolate reductase gene using the nascent strand length assay. Three DNA marker segments with known positions relative to this replication origin were amplified by PCR. By quantitatively assessing the length of DNA the fragments in all fractions containing these markers, the location of the replication origin was established. In either control cells or corn oil treated cells, the location of ori-ß was found to be consistent with previous studies. However, in fish oil treated cells, DNA replication appears to start from a new site located further upstream from ori-ß, suggesting a different replication initiation pattern. This study suggests novel mechanism(s) by which fats affect cell proliferation and DNA replication in mammalian cells.