Klebsiella pneumoniae type 3 fimbriae are encoded by mrkABCDF genes which produce the major pilin subunit MrkA, chaperone MrkB, outer membrane usher MrkC, adhesin MrkD and MrkF of unknown function, respectively. RT-PCR analysis demonstrated that the mrkF gene is contained within the mrk operon. Deletion of mrkF in K. pneumoniae CG43 was found to reduce biofilm formation. A higher level of biofilm formation activity was also observed in recombinant Escherichia coli JM109[pmrkABCDF] compared to that observed for JM109[pmrkABCD]. Immunoelectron microscopy analysis of recombinant type 3 fimbriae using anti-MrkA and anti-MrkF antibody-labeled gold particles revealed that MrkF intermittently inserted into the MrkA filament. An interaction between recombinant MrkA and MrkF was demonstrated by co-immunoprecipitation analysis, further supporting the notion that MrkF is a structural component of the fimbriae. Intriguingly, the incorporation of MrkF appeared to decrease fimbrial length but increased activity of autoaggregation and biofilm formation in the bacteria JM109[pmrkABCDF]. This suggested that MrkF may play a role in assembly of the filament.