The classical concept of the arachidonic and/or linoleic acid cascade comprises the liberation of free polyenoic fatty acids from membrane phospholipids and subsequent oxygenation via the cyclooxygenase or lipoxygenase pathways forming biologically active compounds such as prostaglandins, thromboxane, and leukotrienes. Alternatively, 15- lipoxygenases can catalyze the direct oxygenation of esterified fatty acids within biomembranes. We have also observed that the "leukocyte- type" but not the "platelet-type" 12-lipoxygenase can oxygenate complex ester lipids. The generation of oxidized esterified lipids has been implicated as mediating a variety of physiological and pathological events. In Syrian hamster embryo(SHE) fibroblasts, the mitogenic polypeptide epidermal growth factor(EGF) stimulates the lipoxygenase oxidation of phospholipids. By reverse and straight phase-HPLC, chiral analysis, UV, and GC-MS techniques, we have identified the primary membrane product as 13(S)-hydroxylinoleic acid(HODE). We characterized this compound as a potent enhancer of EGF-dependent DNA synthesis in SHE cells. EGF stimulates HODE incorporation into cellular membranes in a dose- and time-dependent manner. HODE uptake, incorporation, and mobilization is regulated by the intrinsic tyrosine kinase activity of the EGF receptor and this effect is lost in SHE cells that lack tumor suppressor activity(supB~). The level of 13-HODE uptake in cells that retain the tumor suppressor phenotype (supB+) is twice that of supB. Inhibition of tyrosine phosphatase activity with vanadate potentiates HODE uptake in supB+ but not supB~ cells. Moreover, activation of protein kinase C with phorbol ester stimulates HODE incorporation in the supB+ line only. The differential effects of EGF on HODE uptake and mobilization in SHE cells appear to be related to loss of the tumor suppressor phenotype. This biochemical process may be important in determining the mechanism and site of 13-HODE interaction with the EGF mitogenic signaling pathway. These studies should expand current consideration of how mono-hydroxy fatty acids and oxygenated esterified lipids exert their biological actions.