Cultured human skin fibroblasts were incubated in the presence of [14C]arachidonic acid (50 microM; 1 m Ci/mmol) and the divalent cation ionophore A23187 at 37 degrees C for 60 min. The metabolites formed were extracted from the cell-free medium in diethyl ether, separated by thin layer chromatography and identified unequivocally by GC-MS. The distribution of the arachidonic acid metabolites as estimated from the recovered radioactivity showed as major product prostaglandin E2 (26%). Minor amounts of other prostaglandins, i.e., 6-oxo-prostaglandin F1 alpha (1%), prostaglandin F2 alpha (1%), prostaglandin D2 (0.5%) and prostaglandin A2 (1%) were also present. In addition to the prostaglandins, monohydroxy fatty acids (4.5%) were also detected. This fraction contained 33% 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT), 22% 11-hydroxy-5,8,11,14-eicosatetraenoic acid (11-HETE) and 31% 15-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE). Lipid extracts of the cells did not show any detectable amount of the monohydroxy fatty acids, indicating that they are not incorporated metabolically in the cellular lipids. The monohydroxy fatty acids originate mainly from the exogenously added arachidonic acid as evidenced by the 2H/H ratio (30:1) from experiments with octadeuterated arachidonic acid [( 2H8]arachidonic acid). Indomethacin inhibited the formation of all prostaglandins, HHT and 11-HETE; moreover, eicosatetraynoic acid (also blocked the formation of 15-HETE. From these results, it can be concluded that in human skin fibroblasts prostaglandin E2 is the major product of the cycloxygenase pathway, while 15-HETE is the main lipoxygenase product.