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Studies from high mortality areas have suggested that diphtheria-tetanus-pertussis may be associated with an increase in the mortality of girls relative to boys. We therefore examined whether hepatitis B vaccine (HBV) was associated with sex-specific differences in mortality.As part of a randomized trial of measles vaccine, a subcohort of 876 children was offered HBV at 7½, 9 and 10½ months of age. We examined whether this cohort differed in mortality rate and female-male mortality ratio compared with previous and subsequent birth cohort enrolled in the same trial.Four districts in Bissau, the capital of Guinea-Bissau.Six annual birth cohorts of 8906 children registered in the study area and followed from 1½ to 12 months of age between March 1995 and February 2001. Of these children, 6399 took part in a 2-dose measles vaccination trial; of those born between March 1996 and February 1997, 876 received HBV.(1) The mortality rate ratio (MR) between 7½ and 12 months and 1½ and 7½ months old children; (2) the female-male MR among trial children having received HBV plus measles vaccine or only measles vaccine.In cohorts not receiving HBV, the MR for children 7½–12 and 1½-7½ months of age was 0.97 “95% confidence interval (95% CI), 0.79–1.24”, whereas the MR was 1.62 (95% CI 1.09–2.41) in the cohort receiving HBV at 7½ months (test of homogeneity, P = 0.030). Among children enrolled in the measles vaccination trial, HBV-vaccinated children 7½–12 months of age had higher mortality than both prior and subsequent cohorts who had not received HBV (MR = 1.81; 95% CI 1.19–2.75), the difference being particularly strong for girls (MR=2.27; 95% CI 1.31–3.94). In the cohort who had received both HBV and measles vaccine, the female-male MR between 9 and 24 months of age was 2.20 (95% CI 1.07–4.54) compared with 0.96 (95% CI 0.70–1.32) in trial participants who had received measles vaccine only (test for homogeneity, P = 0.040). With longer follow-up, these tendencies remained the same.These comparisons suggested changes in the mortality pattern after the introduction of HBV, particularly for girls. Hence in areas with high mortality, HBV may affect girls’ and boys’ susceptibility to infections differently.