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To identify factors that could explain differences in rate of spread of HIV between different regions in sub-Saharan Africa.Cross-sectional study.The study took place in two cities with a relatively low HIV prevalence (Cotonou, Benin and Yaoundé, Cameroon), and two cities with a high HIV prevalence (Kisumu, Kenya and Ndola, Zambia). In each of these cities, a representative sample was taken of about 1000 men and 1000 women aged 15-49 years. Consenting men and women were interviewed about their socio-demographic background and sexual behaviour; and were tested for HIV, herpes simplex virus type 2 (HSV-2), syphilis, Chlamydia trachomatis and Neisseria gonorrhoea infection, and (women only) Trichomonas vaginalis. Analysis of risk factors for HIV infection was carried out for each city and each sex separately. Adjusted odds ratios (aOR) were obtained by multivariate logistic regression.The prevalence of HIV infection in sexually active men was 3.9% in Cotonou, 4.4% in Yaoundé, 21.1% in Kisumu, and 25.4% in Ndola. For women, the corresponding figures were 4.0, 8.4, 31.6 and 35.1%. High-risk sexual behaviour was not more common in the high HIV prevalence cities than in the low HIV prevalence cities, but HSV-2 infection and lack of circumcision were consistently more prevalent in the high HIV prevalence cities than in the low HIV prevalence cities. In multivariate analysis, the association between HIV infection and sexual behavioural factors was variable across the four cities. Syphilis was associated with HIV infection in Ndola in men [aOR = 2.7, 95% confidence interval (CI) = 1.5-4.9] and in women (aOR = 1.7, 95% CI = 1.1-2.6). HSV-2 infection was strongly associated with HIV infection in all four cities and in both sexes (aOR ranging between 4.4 and 8.0). Circumcision had a strong protective effect against the acquisition of HIV by men in Kisumu (aOR = 0.25, 95% CI = 0.12-0.52). In Ndola, no association was found between circumcision and HIV infection but sample sizes were too small to fully adjust for confounding.The strong association between HIV and HSV-2 and male circumcision, and the distribution of the risk factors, led us to conclude that differences in efficiency of HIV transmission as mediated by biological factors outweigh differences in sexual behaviour in explaining the variation in rate of spread of HIV between the four cities.