The goal of this European pilot study was to evaluate the knowledge, attitudes and beliefs of prison staff from five countries towards HIV infection and to identify factors related to the potential discrimination of HIV-positive inmates. The survey revealed that the levels of knowledge with regard to HIV transmission and the degrees of tolerance varied significantly between prisons. A large proportion of staff overestimated the prevalence of HIV in their prison and feared being contaminated. The willingness of the staff to know the inmates' HIV seropositive status was negatively correlated to their level of tolerance; however, it was positively correlated to their knowledge of the modes of HIV transmission. This study underlines the necessity to improve HIV/AIDS prevention policy for prison staff in order to strengthen good practice in terms of managing the risk of contamination and hindering discrimination.