OBJECTIVES: To estimate HIV incidence and explore evidence for changing sexual behaviour over time among men and women belonging to different socioeconomic groups in rural South Africa. DESIGN AND METHODS: A cohort study conducted between 2001 and 2004; 3881 individuals aged 14-35 years enumerated in eight villages were eligible. At least three household visits were made to contact each eligible respondent at both timepoints. Sexual behaviour data were collected in structured, respondent-focused interviews. HIV serostatus was assessed using an oral fluid enzyme-linked immunosorbent assay at each timepoint. RESULTS: Data on sexual behaviour were available from 1967 individuals at both timepoints. A total of 1286 HIV-negative individuals at baseline contributed to the analysis of incidence. HIV incidence was 2.2/100 person-years among men and 4.9/100 person-years in women, among whom it was highest in the least educated group. Median age at first sex was lower among later birth cohorts. A higher number of previously sexually active individuals reported having multiple partners in the past year in 2004 than 2001. Condom use with non-spousal partners increased from 2001 to 2004. Migrant men more often reported multiple partners. Migrant and more educated individuals of both sexes and women from wealthier households reported higher levels of condom use. DISCUSSION: HIV incidence is high in rural South Africa, particularly among women of low education. Some risky sexual behaviours (early sexual debut, having multiple sexual partners) are becoming more common over time. Condom use is increasing. Existing HIV prevention strategies have only been partly effective in generating population-level behavioural change.