Objectives: (1) To describe the epidemiology of neonatal group B streptococcal (GBS) disease over five years (1997–2001) in the Netherlands, stratified for proven and probable sepsis and for very early (<12 h), late early (12 h – <7 days) and late (7–90 days) onset sepsis. (2) To evaluate the effect of the introduction in January 1999 of guidelines for prevention of early onset GBS disease based on risk factors. Methods: Data on cases were collected in collaboration with the Dutch Paediatric Surveillance Unit and corrected for under-reporting by the capture-recapture technique. Results: Total incidence of proven very early onset, late early onset and late onset GBS sepsis was 0.32, 0.11 and 0.14 per 1000 live births, respectively, and of probable very early onset, late early onset and late onset GBS sepsis was 1.10, 0.18 and 0.02 per 1000 live births, respectively. Maternal risk factors were absent in 46% of the proven early onset cases. Considerably more infants with proven GBS sepsis were boys. 64% of the infants with proven very early onset GBS sepsis were first borns compared with 47% in the general population. After the introduction of guidelines the incidence of proven early onset sepsis decreased considerably from 0.54 per 1000 live births in 1997–8 to 0.36 per 1000 live births in 1999–2001. However, there was no decrease in the incidence of meningitis and the case fatality rate in the first week of life. The incidence of late onset sepsis also remained unchanged. Conclusion: After the introduction prevention guidelines based on risk factors there has been a limited decrease in the incidence of proven early onset GBS sepsis in the Netherlands. This study therefore recommends changing the Dutch GBS prevention guidelines.