The objectives were to study the effect of microbial 70 kDa heat shock protein (HSP70) on the production of tumour necrosis factor (TNF)-α and interleukin (IL)-12 by dendritic cells (DC) from patients with Crohn's disease. TNF-α concentration was increased significantly when DC from Crohn's disease were stimulated with HSP70 or CD40L and this was associated with signalling by the extracellular signal regulated kinase (ERK) 1/2 and p38 mitogen activated protein (MAP) kinase pathway. IL-12 production was also increased when DC were stimulated with HSP70. Cells eluted from inflamed intestinal mucosa from Crohn's disease, stimulated with HSP70, CD40L or lipopolysaccharide produced significantly greater TNF-α and IL-12 concentrations than cells from uninflamed mucosa. Significant inhibition of TNF-α production was demonstrated when DC from peripheral blood mononuclear cells or cells eluted from intestinal mucosa of Crohn's disease were treated with either the HSP70 inhibitory peptide (aa 457–496) or peptides derived from CD40 and CD40L. These inhibitory peptides target the CD40–CD40L and the emerging CD40–HSP70 co-stimulatory pathway. Our findings offer a novel strategy to prevent excessive production of TNF-α in Crohn's disease.