After a single in vivo application of 20 nmol of the 'complete' tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) or of the 'incomplete' promoter 12-O-retinoylphorbol-13-acetate (RPA) to the back skin of mice, a major arachidonic acid metabolite (AAMX) was found in cell-free epidermal preparations after addition of arachidonic acid (AA). Studies with cyclooxygenase and lipoxygenase inhibitors indicated that this metabolite is a lipoxygenase product. The metabolite has been purified by sequential use of t.l.c. and h.p.l.c. and identified as 8-hydroxy-5Z,9E,11Z,14Z-eicosatetraenoic acid (8-HETE) by means of GC-MS and 1H-n.m.r. spectroscopy. To assist in the characterization of AAMX, a reference mixture of 8-HETE and 9-HETE was used for which a complete structural analysis could be performed using one- and two-dimensional 1H-n.m.r. at 500 MHz. The production of 8-HETE has been shown to start with a lag phase of 3 h after TPA treatment and to reach a maximum after 24-48 h. Nonpromoting phorbol esters are 10-fold, the Ca2+-ionophore A 23187 100-fold, less efficient in inducing in vivo the lipoxygenase responsible for 8-HETE production. 3-Day-old neonatal mice cannot be induced to generate 8-HETE in response to TPA, whereas 8-day-old mice show an extremely strong response. Neither primary basal keratinocytes, isolated from TPA-treated mouse epidermis nor TPA-treated epidermal cell lines generate 8-HETE, indicating that the metabolite may not originate from these epithelial cells.