OBJECTIVES: To investigate the hypothesis that mother's use of antibiotics in pregnancy could influence asthma and eczema in early life. STUDY DESIGN: Subjects were included from the Copenhagen Prospective Study on Asthma in Childhood cohort of children born of mothers with asthma (N = 411). Severe asthma exacerbations and eczema were diagnosed by research unit physicians. Replication was sought in children from the Danish National Birth Cohort (N = 30 675). Asthma outcomes were hospitalization and use of inhaled corticosteroids. Eczema was defined by an algorithm developed from cases of clinically verified eczema. All children were followed to age 5 years in a cohort study design. RESULTS: The Copenhagen Prospective Study on Asthma in Childhood data showed increased risk of asthma exacerbation (hazard ratio 1.98 [95% CI 1.08-3.63]) if mothers had used antibiotics during third trimester. The Danish National Birth Cohort confirmed increased risk of asthma hospitalization (hazard ratio 1.17 [1.00-1.36]), and inhaled corticosteroids (1.18 [1.10-1.27]) in the children if mothers used antibiotics any time during pregnancy. In the subgroup of mothers using antibiotics for nonrespiratory infection, the children also had increased risk of asthma. CONCLUSION: We found increased risk of asthma associated with maternal antibiotic use in a clinical study of a birth cohort with increased risk of asthma and replicated this finding in an unselected national birth cohort, and in a subgroup using antibiotics for nonrespiratory infections. This supports a role for bacterial ecology in pre- or perinatal life for the development of asthma.