Bacillus cereus is a Gram-positive, spore-forming bacterium that is frequently identified as the causative agent of food-borne diseases and is also implicated in food spoilage of especially dairy products. The capacity of B. cereus to form biofilms on different substrata is of great concern in the food industry. Not only does biofilm formation cause economic loss by equipment failure, but contamination of food products via biofilm cells also raises safety concerns. Bacterial biofilms have been defined as structmed multicellular communities that form through a complex developmental process. In contrast to Gram-negative bacteria, biofilm formation by Gram-positive bacteria has only recently been examined. Relatively few genes have been identified that are required for these bacteria to form biofilms and little is known about how they coordinate biofilm formation. In order to contribute to the advancement of knowledge regarding the process of biofilm formation in Gram-positive bacteria, the aim of this investigation was essentially to identify and characterize genes involved in B. cereus biofilm formation. To investigate. B. cereus ATCC 14579 was subjected to transposon mutagenesis with the Tn917-LTVI transposon. Screening of a collection of3 500 insertional mutants for the ability to form biofilms at the solid-liquid-air interface of glass surfaces led to the identification of eight biofilm-impaired mutants. Each of the mutants contained a single transposon insertion, and no significant differences were observed in the planktonic growth rate between the B. cereus wild-type and biofilm-impaired mutant strains. The chromosomal transposon insertion in three of the mutants mapped to genes involved in purine biosynthesis (pur A, purC and purL), while the transposon insertion in two other mutants mapped to the ftsE gene and to the promoter region of the motA gene, respectively. In one of the mutants the transposon was located in the intergenic region between two divergently transcribed genes, which encodes a murein hydrolase exporter and nucleoside hydrolase, respectively. In the final two biofilmimpaired mutants the transposon was respectively mapped to genes encoding a putative membrane spanning protein and a putative protein of unknown function. Results obtained by quantitative real-time PCR assays indicated that expression of each of the identified B. cereus ATCC 14579 genes, with the exception of the motA gene, was up-regulated in the biofilm population. In the case of motA, expression of the gene was down-regulated 3.2-fold in the biofilm population and results obtained during the course of this investigation indicated that motility, rather than the presence of flagella, is required for B. cereus biofilm formation. Although this result is in agreement with that reported previously for B. subtilis, none of the other genes identified in this investigation have previously been implicated in biofilm formation by Gram-positive bacteria.