Nitric oxide (NO) has been detected in the exhaled gas of animals and humans. In previous work, investigators have used anesthetized, mechanically ventilated animals to obtain exhaled NO (E(NO)) measurements, which has unclear effects on the levels of E(NO) and does not allow for repeated analysis of E(NO). We sought to measure E(NO) from a single, spontaneously breathing mouse. The mouse was placed in a small Plexiglas chamber and allowed to acclimatize before exhaled gas was collected for E(NO) analysis. Under optimal operating conditions of flow and pressure, the mean concentration of exhaled NO (FE(NO)) of 25 mice was 10.1 +/- 1.0 ppb. The maximal variation of FE(NO) when repeatedly measured daily in individual animals was 2.1 ppb. Administration of L-NAME, a nonselective NOS inhibitor, reduced FE(NO) by 51 +/- 6% (p < 0.01). Intraperitoneally administered lipopolysaccharide induced acute lung injury and increased FE(NO) by 30 +/- 7% (p < 0.05). We have demonstrated that it is possible to noninvasively measure E(NO) from a single, spontaneously breathing mouse. This novel technique provides a stable, reproducible, and responsive measure of E(NO) in mice. This technique will be of use in determining cellular and isoform sources of E(NO), as well as the role of endogenous NO in lung disease.