1. The extent to which the p38 mitogen-activated protein (MAP) kinase and MAP kinase kinase (MKK)-1-signalling pathways regulate the expression of granulocyte/macrophage colony-stimulating factor (GM-CSF) from LPS-stimulated human monocytes has been investigated and compared to the well studied cytokine tumour necrosis factor-alpha (TNF alpha). 2. Lipopolysaccharide (LPS) evoked a concentration-dependent generation of GM-CSF from human monocytes. Temporally, this effect was preceded by an increase in GM-CSF mRNA transcripts and abolished by actinomycin D and cycloheximide. 3. LPS-induced GM-CSF release and mRNA expression were associated with a rapid and time-dependent activation of p38 MAP kinase, ERK-1 and ERK-2. 4. The respective MKK-1 and p38 MAP kinase inhibitors, PD 098059 and SB 203580, maximally suppressed LPS-induced GM-CSF generation by >90%, indicating that both of these signalling cascades co-operate in the generation of this cytokine. 5. Electrophoretic mobility shift assays demonstrated that LPS increased nuclear factor kappa B (NF-kappa B) : DNA binding. SN50, an inhibitor of NF-kappa B translocation, abolished LPS-induced NF-kappaB : DNA binding and the elaboration of TNFalpha, a cytokine known to be regulated by NF-kappaB in monocytes. In contrast, SN50 failed to affect the release of GM-CSF from the same monocyte cultures. 6. Collectively, these results suggest that the generation of GM-CSF by LPS-stimulated human monocytes is regulated in a co-operative fashion by p38 MAP kinase- and MKK-1-dependent signalling pathways independently of the activation of NF-kappa B.