Studies were undertaken to examine the effect of methotrexate (MTX) administered in vivo or added in vitro upon the production of the 5-lipoxygenase (5-LO) metabolites of arachidonic acid (AA) by rat neutrophils. Peptone-induced peritoneal exudate cells were stimulated by A23187 and the cell suspensions assayed for leukotriene B4 (LTB4), the all-trans isomers of LTB4 and 5-hydroxyeicosatetraenoic acid (5-HETE) using high-pressure liquid chromatography. MTX added in vitro to rat cells was weakly inhibitory; however, no inhibition of LTB4 production was seen following in vivo administration of MTX by oral, subcutaneous or intraperitoneal routes. On the basis of these findings, inhibition of 5-LO metabolism does not appear to explain the anti-inflammatory effects of MTX.