In The Netherlands, children with at least one parent born in a hepatitis B virus (HBV) endemic country are offered HBV vaccination within the National Immunization Programme (NIP) since 2003. However, in the eligible group the HBV vaccine coverage is lower than the DPT-IPV-Hib coverage. We therefore conducted a questionnaire survey in order to determine the acceptance of HBV vaccination among parents of eligible children. Given the possibility that universal HBV vaccination will be introduced in the Netherlands, we also assessed the attitude towards universal HBV vaccination among parents whose children are currently not eligible for HBV vaccination. Participants were selected based on the registered vaccination status of their child. Only 13 of 83 parents (16%) within the HBV-eligible group whose child was registered as 'incompletely vaccinated' for HBV reported that they refused a vaccine for their child. Risk factors for HBV refusal were a low risk perception of HBV, a high socioeconomic status and one parent born in The Netherlands. Within the non-eligible group, we found that 9% (95% CI: 3-22%) of the parents whose child was fully vaccinated with DPT-IPV-Hib had a negative attitude towards universal HBV vaccination. Considering the recent recommendation of the Dutch Health Council to introduce universal HBV vaccination, this resistance deserves further attention.