Nose-only exposure is used to study the distribution and toxicity of airborne contaminants. Restraint of animals in nose-only tubes causes stress, but the impact on pulmonary mRNA levels is unknown. Since stress and xenobiotics activate common pathways, we assessed whether nose-only exposure would alter expression of toxicologically relevant genes in the lungs. To identify candidate genes for further analysis, we first interrogated microarray data to examine time-dependent changes in gene expression in air-control animals from a nose-only inhalation study involving male wild-type C57BL/6 mice and transgenic tumor necrosis factor (TNF)-alpha over-expressing littermates. Comparison of transcript levels immediately and 24 h after a single 4-h nose-only exposure to air revealed differential expression of 280 genes (false discovery rate-adjusted, p < .05). Functional analysis revealed enrichment of immune response, apoptosis, and signalling terms, consistent with effects of restraint stress. We then selected a subset of target genes for comparison of naive animals and air-exposed animals from the inhalation study by real-time polymerase chain reaction (PCR). Expression of genes involved in stress (BNIP, sestrin-1, CDKN1A [p21], GADD45 gamma), glucocorticoid-response (GILZ, Sgk), and signal transduction (MAP3K6, C/EBP-delta) was increased as a result of nose-only exposure (p < .05). In contrast, proinflammatory factors (lymphotoxin-beta, chemokine receptor CXCR5) were decreased (p < .05). Immune gene responses observed in wild-type animals were reduced in animals with lung inflammation, indicating that pathological states can modify the response to nose-only exposure. Observed responses may warrant consideration in the evaluation of materials delivered by nose-only inhalation, and suggest that incorporation of naive animals into nose-only studies should be considered as a best practice.