Genetic determinants that co-operate with type 1 pili to mediate invasion were sought for in adherent-invasive Escherichia coli strain LF82 isolated from a patient with Crohn's disease. Two mutants selected for their impaired ability to invade epithelial cells carried insertions of a TnphoA transposon within genes of the flagellar regulon. An isogenic mutant LF82-DeltafliC deleted for the flagellin-encoding gene did not adhere, did not invade and, surprisingly, expressed only a few type 1 pili. Type 1 pili downregulation resulted from a preferential switch towards the off-position of the invertible DNA element located upstream of the fim operon. This was also correlated with a decrease in the flagellar regulator flhDC mRNA levels, suggesting that the transcriptional regulator FlhD2C2 could control type 1 pili expression directly or indirectly. Transformation with a cloned fim operon allowed bypass of the type 1 pili downexpression in the LF82-DeltafliC mutant. Thus, we showed that flagella play a direct role in the adhesion process via active motility. In addition to downregulating type 1 pili expression, flagella also play an undefined role in strain LF82 invasion, which is not restricted to motility or flagellar structure, but could be related to co-ordinate expression of invasive determinants.