Previously, the cross-species, quorum-sensing signal AI-2 has been found to induce biofilm formation 30-fold in E. coli K12 (unpublished) by increasing expression of 67 genes, primarily those associated with chemotaxis, motility, and flagellar synthesis (Biotechnol. Bioeng. 88: 630, 2004), including the specific motility loci qseBC and flhD (Mol. Microbiol. 43: 809, 2002). Differential gene expression has also been used to show b3022 is induced 8-fold in biofilms relative to planktonic cells (Appl. Microbiol. Biotechnol. 64: 515, 2004). In this report we show the b3022 locus, which we have re-named motility quorum sensing (mqsA), regulates motility through the two-component motility regulatory system QseBC. Deletion of mqsA decreased biofilm formation in complex media (74%), minimal media (46%), and complex media supplemented with 0.4% glucose (78%) as well as decreased biomass, substratum coverage, and thickness by 9-, 19-, and 4-fold, respectively, in continuous flow cells. By deleting mqsA, wild-type biofilm architecture was altered from a 54-µm-thick structure with macrocolonies to only a few colonies as visualized by confocal microscopy. MqsA positively regulates expression of qseBC, fliA, and motA since deleting mqsA decreased their transcription by 61-, 2.4-, and 18-fold, respectively, in minimal medium. Biofilm formation was not increased upon the addition of 6.4 µM AI-2 for the isogenic deletions of mqsA, qseBC, fliA, and motA indicating AI-2 induces biofilm formation in E. coli with MqsA as the mediator which regulates QseBC.