Abstract: The effects of anisodamine (654-2) on the metabolism of arachidonic acid (AA) by rat pleural neutrophils were investigated. The major products of metabolism by 5-lipoxygenase (5-LPO), measured by HPLC, were LTB₄ and 5-HETE, whereas the major cyclooxygenase (CO) products measured by HPLC and RIA, were HHT and TXB₂. The formation of all products from endogenous AA by neutrophils was significantly depressed by 654-2 in a dose-dependent manner. At the concentration of 3 × 10^-4 mol/L the formation of LTB₄, 5-HETE, HHT and TXB₂ were decreased by 26, 29, 31 and 25%, respectively. This reduction was more than 40% when the concentration of 654-2 was 1 × 10^-3 mol/L. However, the inhibitory effect was not significant when the concentration of 654-2 was below 5 × 10^-5 mol/L. No effect of 654-2 on metabolism of 1-¹⁴C AA by rat neutrophils was observed. The different effects of 654-2 on the metabolism of endogenous and exogenous AA indicate that the inhibitory effect of 654-2 on formation of both 5-LPO and CO products from endogenous substrate may be due to the reduction of AA release from cell membrane rather than inhibition of 5-LPO and CO activity.