Abstract Background Pre-exposure prophylaxis (PrEP) is an effective HIV prevention strategy. Since 2023, in Italy, PrEP has been subject to reimbursement, making it freely available to individuals who meet specific criteria, including those who engage in substance use during sexual activity (chemsex). In this context, all healthcare professionals (HCPs) in Addiction Departments and Services (ADS) play a crucial role from the intake interview in identifying potential PrEP candidates, providing an essential gateway to services. Thus, this study primarily aimed to explore the knowledge of ADS professionals regarding PrEP. Methods In 2024, an ongoing nationwide cross-sectional study has been conducted in Italy, enrolling a convenience sample of HCPs employed in ADS. The online questionnaire comprised items assessing knowledge regarding PrEP, training on PrEP, and experiences related to daily practice. The primary outcome was the awareness of PrEP existence, while secondary outcomes included knowledge regarding reimbursement. Results Preliminary analyses were conducted based on 269 questionnaires. Participants had a median age of 52 years (IQR=45-59). Physicians represented 26.0% of the sample, followed by nurses (23.0%). Awareness of PrEP stood at 43.5%, while knowledge of the existence of reimbursement conditions was at 29.9%. A vast majority (93.7%) felt inadequately informed, while 97.0% believed HCPs in ADS should receive training. Physicians demonstrated significantly higher awareness of PrEP existence, whereas there were no significant differences in reimbursement knowledge compared with other HCPs. Conclusions Intermediate findings revealed substantial gaps in PrEP awareness and reimbursement knowledge. These results emphasize the critical importance of tailored education and training programs to bridge these gaps and facilitate effective PrEP implementation in diverse healthcare settings, ultimately advancing HIV prevention efforts and promoting equitable access to care. Key messages • Substantial knowledge gaps about PrEP among healthcare professionals working in Addiction Departments and Services in Italy were highlighted. • Effective PrEP implementation hinges on empowering professionals with the necessary expertise to identify and support PrEP candidates, underscoring the critical role of tailored education initiatives.