5‐Lipoxygenase (5‐LO) is the key enzyme in the biosynthesis of proinflammatory leukotrienes. Here, we demonstrate that extracellular signal‐regulated kinases (ERKs) can phosphorylate 5‐LO in vitro. Efficient phosphorylation required the presence of unsaturated fatty acids and was abolished when Ser‐663 was mutated to alanine. In intact HeLa cells stimulated with arachidonic acid (AA), impaired 5‐LO product formation was evident in cells expressing the S663A‐5‐LO mutant compared with cells expressing wild‐type 5‐LO. For Mono Mac 6 cells, priming with phorbol myristate acetate (PMA) before stimulation with ionophore was required for ERK1/2 activation and efficient 5‐LO phosphorylation, in parallel with substantial AA release and 5‐LO product formation. Inhibition of PKC by GF109203x or MEK1/2 by U0126 (or PD98059) abolished the 5‐LO up‐regulation effects of PMA. In contrast, these inhibitors failed to suppress 5‐LO product formation induced by stimuli such as AA plus ionophore, which apparently do not involve the ERK1/2 pathway. Based on inhibitor studies, ERKs are also involved in AA‐stimulated 5‐LO product formation in PMNL, whereas a role for ERKs is not apparent in 5‐LO activation induced by ionophore or cell stress. Finally, the data suggest that ERKs and p38 MAPK‐regulated MAPKAPKs can act in conjunction to stimulate 5‐LO by phosphorylation.