Serum samples from 33,363 healthy people in Tunisia have been tested for serological markers of hepatitis B, C and delta viruses (HBV, HCV and HDV). Hepatitis B surface antigen (HBsAg) was detected in 6.5% of sera. The overall seroprevalence of HBV was 37.5%. Vertical and perinatal transmission of HBV in the first 3 months of life occurred in only 0.4% of 177 mother and child pairs. HBV seroprevalence was 10.7% in infants under 5 years old and increased with age rapidly till 25 years of age and then more slowly in adulthood, reaching 54% for people aged over 40 years. HBsAg seropositivity varied throughout the country, ranging from 3% to 13% with higher prevalences in the south and central-west regions. Overall seroprevalences for HDV and HCV were 17.7% and 0.4%, respectively. HDV superinfection occurred later than HBV and increased with age in parallel with HBV. Overall, HCV and HBV infections had different geographical distributions throughout the country. The study confirmed the high prevalence of HBV infection in Tunisia; it occurs mainly in children and teenagers, and vertical and perinatal transmission of HBV does not appear to be significant. HDV superinfection is quite common in Tunisia and occurs in almost 44% of individuals infected with HBV. In contrast, seroprevalence of HCV in the Tunisian general population was low (0.4%). These results indicate differences in the distribution of the viruses and/or different routes of transmission.