BACKGROUND: Staphylococcus aureus in atopic skin has been associated with exacerbation of eczema. Objectives To investigate a possible association between neonatal colonization with S. aureus and the risk of atopic dermatitis (AD) during the first 3 years of life. MATERIALS AND METHODS: The study participants were 356 children born of mothers with asthma from the Copenhagen Prospective Study on Asthma in Childhood. Swabs from the vestibulum nasi and the perineum were cultured at 1 month and 1 year, from acute eczema, and from parents (vestibulum nasi and pharynx). AD development and severity were monitored prospectively. RESULTS: Of the neonates, 5.3% had positive swabs for S. aureus cultured from the vestibulum nasi (51.3%) and/or the perineum (11.3%). Forty-two per cent developed AD, but without association between colonization with S. aureus at 1 month of age and risk of developing AD at 3 years of age. There was a 70% concordance for S. aureus carriage between neonates and parents. At 1 year of age 11.3% children had swabs positive for S. aureus. Fourteen per cent of children tested at the 1-year visit developed AD after the visit but before 3 years of age, but again, there was no association between colonization with S. aureus and the risk of AD. In children seen at acute visits the severity of AD measured by scoring of atopic dermatitis (SCORAD) was significantly higher in children with a positive culture for S. aureus in lesions. CONCLUSIONS: Colonization with S. aureus at 1 month of age is not associated with an increased risk of developing AD during the first 3 years of life.