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Background: Antenatal Care (ANC) during pregnancy can play an important role in the uptake of evidence-based services vital to the health of women and their infants. Studies report positive effects of ANC on use of facility-based delivery and perinatal mortality. However, most existing studies are limited to cross-sectional surveys with long recall periods, and generally do not include population-based samples. Methods: This study was conducted within the Health and Demographic Surveillance System (HDSS) of the International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b) in Matlab, Bangladesh. The HDSS area is divided into an icddr,b service area (SA) where women and children receive care from icddr,b health facilities, and a government SA where people receive care from government facilities. In 2007, a new Maternal, Neonatal, and Child Health (MNCH) program was initiated in the icddr,b SA that strengthened the ongoing maternal and child health services including ANC. We estimated the association of ANC with facility delivery and perinatal mortality using prospectively collected data from 2005 to 2009. Using a before-after study design, we also determined the role of ANC services on reduction of perinatal mortality between the periods before (2005 - 2006) and after (2008-2009) implementation of the MNCH program. Results: Antenatal care visits were associated with increased facility-based delivery in the icddr,b and government SAs. In the icddr,b SA, the adjusted odds of perinatal mortality was about 2-times higher (odds ratio (OR) 1.91; 95% confidence intervals (CI): 1.50, 2.42) among women who received ?1 ANC compared to women who received ≥3 ANC visits. No such association was observed in the government SA. Controlling for ANC visits substantially reduced the observed effect of the intervention on perinatal mortality (OR 0.64; 95% CI: 0.52, 0.78) to non-significance (OR 0.81; 95% CI: 0.65, 1.01), when comparing cohorts before and after the MNCH program initiation (Sobel test of mediation P < 0.001). Conclusions: ANC visits are associated with increased uptake of facility-based delivery and improved perinatal survival in the icddr,b SA. Further testing of the icddr,b approach to simultaneously improving quality of ANC and facility delivery care is needed in the existing health system in Bangladesh and in other low-income countries to maximize health benefits to mothers and newborns. [ABSTRACT FROM AUTHOR]