We investigated the cytotoxic effect of conjugated trienoic fatty acids on various human tumor cell lines: DLD- 1, colorectal; HepG2, hepatoma; A549, lung; MCF-7, breast; and MKN-7, stomach. Conjugated linoleic acid (CLA) and conjugated linolenic acid were prepared from linoleic acid (18:2, n-6) and alpha-linolenic acid (18:3, n-3), respectively, by treatment with 6.6% or 21% potassium hydroxide. Spectrophotometric readings at 235 nm for the conjugated diene formation, and at 268 nm for the conjugated triene, were confirmed for the respective conjugated fatty acids. In addition, tung oil (Aleurites fordii) fatty acids consisting principally of a conjugated triene (eleostearic acid, approximately 80% of total fatty acids) were prepared using an alkaline saponification procedure. All tumor cells were incubated for 24 h with 5-100 microM of the conjugated fatty acids, and MTT dye reduction was measured to verify the cell viability. Among the conjugated fatty acids examined, conjugated linolenic acid and tung oil fatty acids exhibited the most intense cytotoxic effects on DLD-1, HepG2, A549, MCF-7 and MKN-7 cells, while CLA was not cytotoxic to the tumor cells. These results demonstrate that conjugated trienoic fatty acids are more cytotoxic to human tumor cells than the conjugated dienoic fatty acid, CLA.