Biofilms are structured communities of cells encased in a polymeric matrix and adherent to a surface, interface or each other. We report here that the soil bacterium Bacillus subtilis forms biofilms. By confocal scanning laser microscopy, we observed that B. subtilis adhered to abiotic surfaces and formed a three-dimensional structure ≥ 30 µm in depth. These biofilms appeared to be at least partly encased in an extracellular polysaccharide matrix, as they could be stained with Calcofluor, a polysaccharide-binding dye. To understand the molecular mechanism of biofilm formation, we screened previously characterized mutants for a defect in biofilm formation. We found that mutations in spo0A, which encodes the major early sporulation transcription factor, caused a defect in biofilm formation. spo0A mutant cells adhered to a surface in a monolayer of cells rather than a three-dimensional biofilm. The requirement of Spo0A for biofilm development appears to result from its role in negatively regulating AbrB. Mutations in abrB suppressed the biofilm defect of a spo0A mutant, indicating that AbrB negatively regulates at least one gene that is required for the transition from a monolayer of attached cells to a mature biofilm. Implications of biofilm development for the ecology of B. subtilis are discussed.