The ability to augment monocyte functions such as TNF-α-producing capacities confers a high immunostimulating potential to GM-CSF. In the present investigation, the mechanism of the GM-CSF-mediated enhancement of monocyte cytokine production was analysed with regard to the involvement of intracellular signalling pathways. GM-CSF primes human monocytes dose- and time-dependently for enhanced LPS-stimulated TNF-α synthesis. Pre-incubation with 10 ng/ml GM-CSF for 6 h before LPS stimulation (10 ng/ml) caused a 3.4 ± 1.9-fold increase in TNF-α release compared to unprimed controls. This was associated with increased phosphorylation of IκBα and elevated nuclear levels of the NF-κB components p50 and p65 and NF-κB binding to DNA. LPS-induced AP-1 binding to DNA was also enhanced in GM-CSF-pre-incubated cells. GM-CSF treatment also caused a slight increase in TLR4 expression on monocytes while CD14 expression remained unchanged. GM-CSF-priming was unaffected by inhibitors of p38 MAPK (SB203580) and lipoxygenase (NDGA). In contrast, the broad-spectrum tyrosine kinase inhibitor genistein and the MEK-1 inhibitor (PD98059) abrogated GM-CSF priming of TNF-α release and activation of both NF-κB and AP-1. It is concluded that a tyrosine kinase of the GM-CSF-triggered ERK1/2 pathway augments the LPS-induced NF-κB and AP-1 activation.