Mother-to-child transmission of hepatitis B virus (HBV) continues to occur despite immunoprophylaxis. We examined maternal factors contributing to transmission in infants receiving adequate immunoprophylaxis in Alberta, Canada. Prenatal specimens from HBsAg-positive women whose babies developed HBV infection despite immunoprophylaxis (cases) and HBsAg-positive mothers whose babies did not (controls) were tested for HBsAg, HBeAg and HBV DNA. Specimens with detectable DNA underwent HBV genotyping. Routinely collected surveillance data and laboratory test results were compared between cases and controls. Twelve cases and 52 controls were selected from a provincial registry from 2000 to 2005. At the time of prenatal screening, median maternal age was 31 years [interquartile range (IQR): 27.5-34.5], and median gestational age was 12 weeks (IQR 10.0-15.5). Cases were more likely than controls to test positive for HBeAg (77.8% vs. 23.1%; P < 0.05). Of all mothers with detectable viral load (n = 51), cases had a significantly higher median viral load than did controls (5.6 × 10(8)  IU/mL vs. 1750 IU/mL, P < 0.0001). Of the two cases who were HBeAg negative, one had an undetectable viral load 8 months prior to delivery and a sP120T mutation. The viral load in the other case was 14,000 IU/mL. The majority of isolates were genotype B (31.3%) and C (31.3%) with no significant differences in genotype between cases or controls. In this case-control study, transmission of HBV to infants was more likely to occur in mothers positive for HBeAg and with high HBV DNA.