INTRODUCTION: We explored factors associated with differential HIV incidence among women participating in a HIV prevention trial in Harare, Durban, and Johannesburg. The trial had shown no effect of the intervention (diaphragm and lubricant gel) on HIV incidence. METHODS: A prospective cohort analysis was conducted of trial participants followed for 12-24 months. Sociodemographic, biological, and behavioral data were collected at baseline and at quarterly visits. Factors associated with HIV incidence were estimated using multivariable Cox regression models, stratified by study location. Attributable risk was calculated from the adjusted hazard ratios (AHR). RESULTS: There were 309 incident HIV infections among the 4948 women in the analysis. HIV incidence was highest in Durban [6.75/100 person-years; 95% confidence interval (CI): 5.74 to 7.93], lower in Johannesburg (3.33/100 person-years; 95% CI: 2.51 to 4.44), and lowest in Harare (2.72/100 person-years; 95% CI: 2.26 to 3.26). Sexually transmitted infections were important risk factors in Harare [prevalent herpes simplex virus type 2 (HSV2) AHR = 2.56, 95% CI: 1.61 to 4.06; incident HSV2 AHR = 12.60, 95% CI: 2.13 to 21.87; Neisseria gonorrhoeae AHR = 6.82, 95% CI: 2.13 to 21.87] and in Durban (prevalent HSV2 AHR = 1.64, 95% CI: 1.07 to 2.51; N. gonorrhoeae AHR = 4.40, 95% CI: 2.07 to 9.39). In Durban, having multiple partners (adjusted odds ratio (AOR) = 1.78 95% CI: 1.11 to 2.85) and sex although a partner was under the influence of alcohol/drugs (AOR = 1.51 95% CI: 1.05 to 2.16) significantly increased risk, whereas in Johannesburg, sexual debut <16 years (AOR = 2.60 95% CI: 1.30 to 5.17) was a strong predictor of HIV acquisition. DISCUSSION: Important differences were seen in drivers of HIV incidence at the 3 study locations. Results from this analysis imply that targeted HIV programing could have a large impact on incident HIV infection in women, and that the most effective approach will likely vary based on knowledge of the local situation/epidemiology.