The prevalence of hepatitis B surface antigen (HBsAg) was determined in a community-based, cross-sectional, age-stratified sample of children from 0 to 6 years of age (n = 2,299) from the Eastern Cape Province of South Africa. The purpose of the study was to investigate the epidemiology and the age of acquisition of hepatitis B virus (HBV) infection in children, thus providing a preimmunization baseline measure of this infection in the population targeted for HBV immunization in South Africa. Overall, 10.4% (95% CI, 9.2-11.7) of the children tested were HBsAg-positive. There was a high rate of positivity in the 0-6- and 7-12-month age groups at 8.1% (95% CI, 5.5-11.7) and 8.9% (95% CI, 6.1-12.7), respectively, suggesting a higher rate of early acquisition of this infection than previously reported in South Africa. The proportion of HBsAg-positive children increased significantly with increasing age (chi2trend = 5.9, df = 1, P = 0.02), reaching 15.7% in the 61-72-month age group. This is the highest rate of HBV infection reported in community-based children from South Africa, indicating a significant burden of this infection. The difference in HBsAg prevalence between urban and rural children was not statistically significant (chi2 = 0.32, df = 1, P = 0.57). There was also no difference in positivity between males (10.5%; 95% CI, 8.7-12.5) and females (9.8%; 95% CI, 8.1-11.7), (chi2 = 0.006, df = 1, P = 0.94). This study provides the most recent preimmunization, community-based baseline investigation of the epidemiology of HBV infection in children targeted for universal immunization in South Africa.