The impact of commensal bacteria on lymphocyte responses in the upper airways was studied in rat nasal mucosa after infection with the pathogen Mycoplasma pulmonis. Phenotyping was performed in situ by paired immunofluorescence staining in germ-free (GF) and conventional (CV) rats before and 3 wk after the monoinfection. Intraepithelial lymphocytes had expanded significantly in GF (P = 0.02) but not in CV rats. Furthermore, a striking proportional increase of T-cell receptor (TCR)alphabeta(+)CD4(+) cells was observed both in the lamina propria and epithelium of GF (P < 0.01) but not of CV rats. Notably, in contrast to the pre-infection state, both mucosal compartments showed a percentage of TCRalphabeta(+)CD4(+) cells that was significantly higher in GF (P = 0.03-P < 0.01) than in CV rats after the monoinfection. In parallel, both compartments displayed a percentage of TCRalphabeta(+) CD8(+) cells that was decreased in GF (P < 0.01) but not in CV rats. The small fraction of TCRgammadelta(+) T cells observed (< 5%) did not change quantitatively or phenotypically after infection. The size of organized nose-associated lymphoid tissue was, on average, increased 5.2-fold in GF rats versus 2.6-fold in CV rats. Collectively, our results demonstrated that the normal microbiota modulated markedly the nasal immune response elicited by monoinfection with M. pulmonis.