Asymptomatic colonization of the nasopharynx by Streptococcus pneumoniae precedes pneumococcal disease, yet pneumococcal colonization factors remain poorly understood. Many bacterial infections involve biofilms which protect bacteria from host defenses and antibiotics. To gain insight into the genetics of biofilm formation by S. pneumoniae, we conducted an in vitro screen for biofilm-altered mutants with the serotype 4 clinical isolate TIGR4. In a first screen of 6,000 mariner transposon mutants, we repeatedly isolated biofilm-overproducing acapsular mutants, suggesting that the capsule was antagonistic to biofilm formation. Therefore, we screened 6,500 additional transposon mutants in an S. pneumoniae acapsular background. Following this approach, we isolated 69 insertions in 49 different genes. The collection of mutants includes genes encoding bona fide and putative choline binding proteins, adhesins, synthases of membrane and cell wall components, extracellular and cell wall proteases, efflux pumps, ABC and PTS transporters, and transcriptional regulators, as well as several conserved and novel hypothetical proteins. Interestingly, while four insertions mapped to rrgA, encoding a subunit of a recently described surface pilus, rrgB and rrgC (encoding the other two pilus subunits) mutants had no biofilm defects, implicating the RrgA adhesin but not the pilus structure per se in biofilm formation. To correlate our findings to the process of colonization, we transferred a set of 29 mutations into the wild-type encapsulated strain and then tested the fitness of the mutants in vivo. Strikingly, we found that 23 of these mutants were impaired for nasopharyngeal colonization, thus establishing a link between biofilm formation and colonization.The life cycle of obligate commensal and pathogenic bacteria depends on efficient host colonization and transmission. It is increasingly being recognized that bacteria alternate between planktonic and sessile forms of growth, the latter in the form of surface-adherent biofilms-typically, complex microbial communities sometimes comprised of several species living in symbiotic relationships within a structured extracellular matrix of proteins, polysaccharides, and DNA. To form biofilms on a surface, bacteria rely on multiple genetic systems, including those involving attachment, intercellular interactions (e.g., quorum sensing), chemotaxis, carbon sensing, and stress responses (21,23,67,87). Sessile bacteria differ strikingly physiologically and metabolically from their planktonic counterparts, and the establishment of bacterial biofilms on host tissues is thought to lead to expression of fitness determinants, protect against host defenses, and enhance resistance to antibiotics (29, 67). These observations and the fact that over half of all bacterial infections are believed to involve biofilms make the study of the role of biofilms in host-pathogen interactions an area of major scientific and clinical relevance (29,69).Streptococcus pneumoniae frequently colonizes the human oronas...