Abstract Stimulation of human polymorphonuclear leukocytes (PMNL) with granulocyte-macrophage CSF (GM-CSF) results in the enhanced expression of several genes, including some coding for cytokines and enzymes. In this study, we investigated the ability of GM-CSF to up-regulate the human neutrophil 5-lipoxygenase (5-LO), a key enzyme in the leukotriene synthetic pathway. GM-CSF induced a dose- and time-dependent de novo synthesis of the 5-LO in PMNL, as determined by immunoprecipitation of 35S-methionine-labeled 5-LO. This up-regulation occurred within 30 min of treatment with GM-CSF and was observed using concentrations of GM-CSF as low as 30 pM. Prior treatment of the cells with the protein synthesis inhibitor cycloheximide abolished this effect of GM-CSF. Western blot analyses demonstrated that levels of 5-LO did not vary over a 6-h period in unstimulated PMNL treated with CX, and that GM-CSF induced a rapid increase in the total cellular level of 5-LO protein; taken together these results indicated a translational effect of GM-CSF on the expression of the 5-LO. However, GM-CSF did not significantly affect the level of 5-LO mRNA in neutrophils, as determined by Northern blot analysis. Furthermore GM-CSF did not alter the stability of 5-LO mRNA, in agreement with a posttranscriptional effect of GM-CSF on 5-LO expression in PMNL. These results show that human PMNL are capable of up-regulating the expression of the 5-LO in response to physiologic activation.