The colonization of the gastrointestinal tract of patients by the opportunistic gram-negative bacillus Klebsiella pneumoniae generally occurs prior to the development of nosocomial infections. Mutant strain C-81 was isolated owing to its reduced capacity to colonize the digestive tract in a murine model following transposon mutagenesis (N. Maroncle, D. Balestrino, C. Rich, and C. Forestier, Infect. Immun. 70:4729-4734, 2002). Nucleotide sequence analysis showed that the transposon had inserted into the first open reading frame, eefA, of a three-gene locus (eefABC) whose homologue encodes a tripartite efflux pump in Enterobacter aerogenes (M. Masi, J. M. Pages, C. Villard, and E. Pradel, J. Bacteriol. 187:3894-3897, 2005), and this operon includes an additional short (183-bp) potential open reading frame, eefX, upstream of eefA. In vivo assays showed that a DeltaeefA isogenic mutant strain normally colonized the gastrointestinal tract in single-strain tests but was significantly impaired in competition against wild-type strain LM21. Although the cecum was the compartment with the highest number of CFU, the DeltaeefA mutant also was detected in the stomach in numbers smaller than those of the wild-type strain. The expression of this potential efflux pump could not be linked to any antimicrobial drug resistance phenotype, but it conferred on the bacteria an acid tolerance response to inorganic acid. The expression of the eef promoter region, measured via a lacZ reporter construction, was slightly induced by an acidic environment and also by hyperosmolarity but not by the presence of bile salts. These results suggest that an efflux pump can confer measurable ecological benefits on K. pneumoniae in an environment with high competition potential.