ABSTRACT The lpf fimbrial operon oscillates between phase ON and phase OFF expression states, thereby generating heterogeneity within S. enterica serotype Typhimurium populations with regard to expression of long polar fimbrial antigens. To determine whether the proportion of lpf phase variants changes with growth conditions, the lpf phase ON content of cultures was determined after in vitro and in vivo passage. After passage in Luria-Bertani (LB) broth for 120 generations, 96% of cells in a serotype Typhimurium culture carried the lpf operon in the phase ON expression state, regardless of the phase ON/OFF ratio in the inoculum. In contrast, a culture passaged on LB agar plates for 500 generations contained approximately 2% lpf phase ON cells. Differences in the lpf phase ON content of cultures passaged in broth and on plates were not caused by an outgrowth of lpf phase ON or lpf phase OFF cells, since deletion of lpf biosynthesis genes did not alter the phase ON/OFF ratio attained after passage. Instead, growth in LB broth resulted in a eightfold increase in the phase OFF-to-ON transition frequency and a decrease of the lpf phase ON-to-OFF transition frequency by a factor of 150 compared to growth on LB agar plates. After infection of naïve CBA/J mice with an lpf phase ON culture of serotype Typhimurium, the proportion of lpf phase ON cells continuously decreased over time, regardless of whether the strain carried intact fimbrial biosynthesis genes. These data suggest that elaboration of fimbriae does not have a major influence on the population heterogeneity produced by phase variation of the lpf operon in naïve mice.