Platelets metabolize 7,10,13,16,19-docosapentaenoic acid (22:5(n-3] into 11-hydroxy-7,9,13,16,19- and 14-hydroxy-7,10,12,16,19-docosapentaenoic acid via an indomethacin-insensitive pathway. Time-dependent studies with 20 microM substrate show a lag in the synthesis of both the 11- and 14-isomers which was not observed for the synthesis of thromboxane B2 (TXB2), 5,8,10-heptadecatrienoic acid, and 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) from arachidonic acid. When platelets were incubated with increasing concentrations of 22:5(n-3), the 11- and 14-isomers were not produced until the substrate concentration exceeded 5 microM unless arachidonic acid was also added to the incubations. The stimulatory effect of arachidonic acid was not blocked by indomethacin thus suggesting that 12-hydroperoxyeicosatetraenoic acid or 12-HETE derived from arachidonic acid may activate the platelet lipoxygenase(s) which metabolize 22:5(n-3). Incubations containing 20 microM 22:5(n-3) and increasing levels of [1-14C]arachidonic acid show that the (n-3) acid inhibits the synthesis of both 5,8,10-heptadecatrienoic acid and TXB2 from arachidonic acid. At the same time, 12-HETE synthesis increased due to substrate shunting to the lipoxygenase pathway.