From the invasive Citrobacter freundii strain 3009, an invasion determinant was cloned, sequenced, and expressed. Sequence analysis of the determinant showed high homology with the fim determinant from Salmonella enterica serovar Typhimurium. The genes of the invasion determinant directed invasion of recombinant Escherichia coli K-12 strains into human epithelial cell lines of the bladder and gut as well as mannose-sensitive yeast agglutination and were termed fim Cf genes. Expression of the Fim Cf proteins was shown by 35 S labeling and/or Western blotting. In the infant rat model of experimental hematogenous meningitis, C. freundii strain 3009 and the in vitro invasive recombinant E. coli K-12 strain harboring the fim Cf determinant reached the cerebrospinal fluid, in contrast to the case for the control strain. The fim determinant was also necessary for efficient in vitro invasion by C. freundii, because a deletion mutant was strongly reduced in its invasion efficiency. The mutation could be complemented in trans by the corresponding genes. Invasion by C. freundii could be blocked only by D-mannose, GlcNAc, and chitin hydrolysate and not by other carbohydrates tested. In contrast, yeast agglutination was not affected by GlcNAc or chitin hydrolysate. This finding indicated mannose residues to be essential for both yeast agglutination and invasion, whereas GlcNAc (oligomer) residues of host cells are involved exclusively in invasion. These results showed the fim determinant of C. freundii to be responsible for D-mannose-and GlcNAc-dependent in vitro invasion without being assembled into pili and for crossing of the blood-brain barrier in the infant rat model.