This article seeks to evaluate knowledge, practices and attitudes of health care workers (HCW) involved in antenatal care in the Unified Health System (SUS) in Rio de Janeiro City (RJC) and to identify major barriers to the implementation of treatment for syphilis in pregnancy care protocols. A cross-sectional study was conducted with 102 HCW in antenatal care at SUS, corresponding to 70% of the eligible pool. Univariate and bivariate analysis were performed using SPSS version 16.0. A number of barriers were identified with respect to knowledge of and familiarity with the current protocols, difficulties related to DST management, relationship with patients and clinics organizational context, which were distinct according to the type of health unit. HCW who had greater access to training and technical manuals had a better performance, although the overall effect was discrete. Identifying barriers to adherence to health care protocols is essential to formulate intervention strategies. Access to protocols through training and technical manuals showed a discrete effect in the improvement of the care delivered to patients, pointing to the need of innovative ongoing education of HCW.