In vitro techniques make a major contribution to the development of alternatives to the in vivo "Draize" skin irritation test, and the development of sensitive and generally applicable in vitro endpoints of cutaneous toxicity is an area of intensive research. To investigate in vitro characteristics of cutaneous irritation, skin explants of rabbit and human origin were topically exposed to chemical irritants, after which the culture medium was analyzed for the presence of metabolites of both arachidonic and linoleic acid. In rabbits exposed to the potent irritant benzalkonium chloride, a direct relation was established between clinical signs of irritation and in vitro release of the proinflammatory mediator 12-hydroxyeicosatetraenoic acid (12-HETE) by the exposed skin. Histological examination revealed varying degrees of epidermal damage. 12-HETE was also the predominant hydroxy fatty acid released in a dose-dependent way by rabbit skin cultures after in vitro exposure to sodium dodecyl sulfate (SDS), benzalkonium chloride (BC), and formaldehyde (FA). Human skin cultures released, in addition to 12-HETE, predominantly 15-HETE and 13-hydroxyoctadecadienoic acid (13-HODE), omega-6 oxygenase products of arachidonic acid and linoleic acid, respectively. The irritant-induced release of hydroxy fatty acids was strongly inhibited by the lipoxygenase inhibitor eicosatetraynoic acid, indicating enzyme-mediated generation of these bioactive lipids. Comparison of hydroxy fatty acid release to more established markers of cytotoxicity (leakage of the cellular enzymes, such as aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH)) revealed that increased levels of 13-HODE, 9-HODE, 12-HETE, and ALT were specific markers of cutaneous irritancy in rabbit skin cultures.