Bacteria undergo significant changes during adherence to surfaces and biofilm development. Cell-to-cell signalling molecules are known to be involved in these phenotypic adaptations to the sessile mode of life. We demonstrated previously that indole can act as an extracellular signal to regulate biofilm formation in E. coli. To identify proteins over- or under-expressed in response to E. coli biofilm formation and indole signalling, we compared the proteomes of the E. coli S17-1 wild-type and 3714 (S17-1 tnaA::Tn5) tryptophanase-negative mutant cells (which don't produce indole) grown as suspensions or biofilms in the presence or absence of exogenous indole. From computer-assisted image analysis, 407 spots were discriminated on two-dimensional electropherograms. Principal component analysis (PCA) of the electropherograms did not discriminate between the proteomes of the wild-type and mutant cells grown as suspensions indicating that indole has a limited impact onto protein expression of planktonic cells. The first principal component extracted by PCA, after standardization of the observations, opposed planktonic and biofilm cells confirming the existence of changes in protein expression during E. coli biofilm formation. Among proteins over- or under-expressed by both sessile wild-type and mutant cells, we identified metabolic enzymes, transporters, proteins involved in the translation and transcription machinery, stress response and regulation, and signalling proteins. The wild-type and mutant strains grown as biofilms in the presence of indole were discriminated by the second component. The role of some proteins whose expression was altered in biofilm bacteria compared to suspended counterparts is discussed.