Human obesity is characterised by an increase in both the number of adipocytes and by an increase in their size, which is determined by adipogenesis. Adipogenesis can be induced in vitro in preadipocytes cultured in differentiation medium containing 3-isobutyl-1-methylxanthine, insulin, dexamethasone, and triiodothyronine but the signalling pathways important in adipogenesis are not well characterised. The p38 mitogen-activated protein kinase (MAPK) signalling pathway is usually associated with stress-activated signalling pathways, but is also important in TNFalpha signalling pathways. TNFalpha induces adipocyte dedifferentiation and apoptosis. Using western blotting, we showed that TNFalpha rapidly increased p38 MAPK phosphorylation in human preadipocytes and differentiated preadipocytes. An inhibitor of p38 MAPK, SB203580 was used to determine effects on human adipocyte differentiation. Primary human preadipocytes were treated with SB203580 (10-6M) and induced to differentiate over 20 days. Differentiation was assessed by measuring the leptin, lipid, protein content, and lipogenic activity. Each mean value (n=4) is given as a percentage of control and the lipid content and lipogenesis data have been corrected for protein content. Protein content was not significantly inhibited with SB203580 treatment (88.17 plus/minus 3.71). Lipid content measured by oil red O staining was inhibited with SB203580 treatment (74.45 plus/minus 5.44 p<0.05) and so was lipogenesis measured by the uptake of 14C-labelled glucose at the end of differentiation (36.68 plus/minus 4.97 p<0.01). Leptin secretion (n=3) measured over 12 days was significantly inhibited with SB203580 treatment compared to the control (Control 10.52 plus/minus 3.09 ng/ml : SB203580 0.67 plus/minus 0.54 ng/ml p<0.01). We conclude that the p38 MAPK pathway is essential for adipocyte differentiation and TNFalpha does not exert its dedifferentiating effects by the p38 MAPK signalling pathway.