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 three weeks after the monoinfection. Intraepithelial lymphocytes had expanded significantly in GF (p=0.02) but not CV rats. Furthermore, a striking proportional increase of T-cell receptor (TCR)⁺CD4⁺ cells was observed both in the lamina propria and epithelium of GF (p<0.01) but not CV rats. Notably, in contrast to the pre-infection state, both mucosal compartments showed a percentage of TCR⁺CD4⁺ cells that was significantly higher in GF (p=0.03-p<0.01) than CV rats following the monoinfection. In parallel, both compartments displayed a percentage of TCR⁺CD8⁺ cells that was decreased in GF (p<0.01) but not in CV rats. The small fraction of TCR⁺ 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 vs. 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.