Currently South Africa does not have national HIV incidence data based on laboratory testing of blood specimens. The 2005 South African national HIV household survey was analysed to generate national incidence estimates stratified by age, sex, race, province and locality type, to compare the HIV incidence and HIV prevalence profiles by sex, and to examine the relationship between HIV prevalence, HIV incidence and associated risk factors. The detection of recent infections was performed on confirmed HIV-positive samples, using the BED capture enzyme immunoassay optimised for dried blood spot (DBS) specimens. BED HIV incidence calculations applied adjustment procedures that were recently revised and approved by the Centers for Disease Control and Prevention for subtype C blood specimens. HIV incidence in the study population aged 2 years and older was 1.4% per year, with 571,000 new HIV infections estimated for 2005. An HIV incidence rate of 2.4% was recorded for the age group 15-49 years. The incidence of HIV among females peaked in the 20-29-year age group at 5.6%, more than six times the incidence found in 20-29-year-old males (0.9%). Among youth aged 15-24 years, females account for 90% of the recent HIV infections. Non-condom use among youth, current pregnancy and widowhood were the socio-behavioural factors associated with the highest HIV incidence rates. The HIV incidence estimates reflect the underlying transmission dynamics that are currently at work in South Africa. The findings suggest that the current prevention campaigns are not having the desired impact, particularly among young women.