Analysis of the temporal development of Escherichia coli K-12 biofilms in complex medium indicates the greatest differential gene expression between biofilm and suspension cells occurred in young biofilms at 4 and 7 h (versus 15 and 24 h). The main classes of genes differentially expressed (biofilm versus biofilm and biofilm versus suspension cells) include 42 related to stress response (e.g. cspABFGI), 66 related to quorum sensing (e.g. ydgG, gadABC, hdeABD), 20 related to motility (e.g. flgBCEFH, fliLMQR, motB), 13 related to fimbriae (e.g. sfmCHM, fimZ, csgC), 24 related to sulfur and tryptophan metabolism (e.g. trpLBA, tnaLA, cysDNCJH), 80 related to transport (e.g. gatABC, agaBC, ycjJ, ydfJ, phoU, phnCJKM), and six related to extracellular matrix (e.g. wcaBDEC). Of the 93 mutants identified and studied, 76 showed altered biofilm formation. Biofilm architecture changed from thin and dense to globular and dispersed to dense and smooth. The quorum-sensing signal AI-2 controls gene expression most clearly in mature biofilms (24 h) when intracellular AI-2 levels are highest. Sulfate transport and metabolism genes (cysAUWDN) and genes with unknown functions (ymgABCZ) were repressed in young (4, 7 h) biofilms, induced in developed biofilms (15 h), and repressed in mature (24 h) biofilms. Genes related to both motility and fimbriae were induced in biofilms at all sampling time points and colanic acid genes were induced in mature biofilms (24 h). Genes related to dihydroxyacetone phosphate synthesis from galactitol and galactosamine (e.g. gatZABCDR, agaBCY) were highly regulated in biofilms. Genes involved in the biosynthesis of indole and sulfide (tnaLA) are repressed in biofilms after 7 h (corroborated by decreasing intracellular indole concentrations in biofilms). Cold-shock protein transcriptional regulators (cspABFGI) appear to be positive biofilm regulators, and deletions in respiratory genes (e.g. hyaACD, hyfCG, appC, narG) increased biofilm formation sevenfold.