E-selectin is an adhesion molecule transiently and specifically expressed on endothelial cells upon stimulation with cytokines. We wished to determine whether methylation could play a role in cell-type specific expression of this gene. We found that the E-selectin promoter in cultured endothelial cells is under-methylated in comparison with non-expressing HeLa cells. Plasmid constructs carrying a reporter driven by the E-selectin promoter and methylated in vitro are no longer transcribed in either an in vitro transcription system or in transiently transfected cells. We identified the NF-kappa B site in the promoter as the likely target for this methylation-mediated repression by testing a minimal promoter carrying only this and an associated element. We conclude that methylation is likely to play a role in blocking E-selectin expression in non-endothelial cells.