The effect of pre- and post-natal smoke exposure on exhaled nitric oxide fraction (F(eNO)) in infants was evaluated and the association between respiratory symptoms and F(eNO) in the first 2 months of life was investigated. The Generation R study is a population-based, prenatally recruited birth cohort. Exposures were assessed by means of questionnaires prospectively administered during pregnancy and after birth. Successful off-line F(eNO) measurements during tidal breathing were obtained in 187 infants (median age 6.9 weeks). The association between possible determinants and log F(eNO) was investigated with multiple linear regression analysis. Infants exposed pre- and post-natally to smoke showed lower F(eNO) than infants exposed only after birth (geometric mean difference (95% confidence interval) 1.5 (1.0-2.1) ppb) and never-exposed infants (1.4 (1.0-1.8) ppb). F(eNO) was reduced in infants with severe upper respiratory symptoms compared with infants with nonsevere symptoms (1.6 (1.0-2.4) ppb). Infants with symptoms of the lower respiratory tract had lower F(eNO) than asymptomatic infants (1.2 (1.0-1.50) ppb). In conclusion, the nature of the association between smoke exposure and exhaled nitric oxide fraction is dependent on timing and intensity of exposure. The occurrence and the severity of respiratory symptoms in the first 2 months of life are associated with lower exhaled nitric oxide fraction.