Juniperonic acid (Delta-5c,11c,14c,17c-20:4, JA) is a polymethylene-interrupted (PMI) fatty acid that occurs in Biota orientalis. In this study, we found that JA has an antiproliferative activity. Swiss 3T3 cells were preloaded with fatty acids before stimulation with bombesin, a mitogenic neuropeptide, and proliferation of the cells was assessed by [(3)H]thymidine incorporation. Preloading of linoleic acid (Delta-9c,12c-18:2) significantly enhanced bombesin-induced proliferation. In contrast, preloading of eicosapentaenoic acid (Delta-5c,8c,11c,14c,17c-20:5, EPA) suppressed proliferation. Likewise, cells preloaded with JA showed a significantly curtailed response to bombesin. The antiproliferative potency of JA was equivalent to that of EPA. Sciadonic acid (Delta-5c,11c,14c-20:3), an omega-6 analogue of JA did not show antiproliferative activity, suggesting the importance of the omega-3 double bond rather than the PMI structure. The EPA-like activity of JA may be involved in the pharmaceutical activity of biota seeds, a psychoactive Chinese traditional medicine.