Uropathogenic Escherichia coli (UPEC) is the primary cause of urinary tract infection (UTI) in the developed world. The major factors associated with virulence of UPEC are fimbrial adhesins, which mediate attachment to specific receptors, enhance persistence and trigger innate host responses. UPEC produce a range of fimbrial adhesins, with type 1 and P fimbriae of the chaperone-usher subclass being the best characterized. The prototype UPEC strain CFT073 contains ten gene clusters that contain genes characteristic of this class of fimbriae. However, only five of these gene clusters have been characterized in detail. In this study the F9 fimbrial gene cluster (c1931-c1936) from CFT073 has been characterized. The F9 fimbriae-encoding genes were PCR amplified, cloned and expressed in a K-12 background devoid of type 1 fimbriae. While F9 fimbrial expression was not associated with any haemagglutination or cellular adherence properties, a role in biofilm formation was observed. E. coli K-12 cells expressing F9 fimbriae produced a dense and uniform biofilm in both microtitre plate and continuous-flow biofilm model systems. In wild-type UPEC CIFT073, expression of the F9 major subunit-encoding gene was detected during exponential growth in M9 minimal medium. F9 expression could also be detected following selection and enrichment for pellicle growth in a CIFT073fim foc double mutant. The F9 genes appear to be common in UPEC and other types of pathogenic E coli. However, their precise contribution to disease remains to be determined.