OBJECTIVES: To assess bias in estimates of STD prevalence in population based surveys resulting from diagnostic error and selection bias. To evaluate the effects of such biases on STD prevalence estimates from three community randomised trials of STD treatment for HIV prevention in Masaka and Rakai, Uganda and Mwanza, Tanzania. METHODS: Age and sex stratified prevalences of gonorrhoea, chlamydia, syphilis, HSV-2 infection, and trichomoniasis observed at baseline in the three trials were adjusted for sensitivity and specificity of diagnostic tests and for sample selection criteria. RESULTS: STD prevalences were underestimated in all three populations because of diagnostic errors and selection bias. After adjustment, gonorrhoea prevalence was higher in men and women in Mwanza (2.8% and 2.3%) compared to Rakai (1.1% and 1.9%) and Masaka (0.9% and 1.8%). Chlamydia prevalence was higher in women in Mwanza (13.0%) compared to Rakai (3.2%) and Masaka (1.6%) but similar in men (2.3% in Mwanza, 2.7% in Rakai, and 2.2% in Masaka). Prevalence of trichomoniasis was higher in women in Mwanza compared to women in Rakai (41.9% versus 30.8%). Herpes simplex virus type 2 (HSV-2) seroprevalence and prevalence of serological syphilis (TPHA+/RPR+) were similar in the three populations but the prevalence of high titre syphilis (TPHA+/RPR >/=1:8) in men and women was higher in Mwanza (5.6% and 6.3%) than in Rakai (2.3% and 1.4%) and Masaka (1.2% and 0.7%). CONCLUSIONS: Limited sensitivity of diagnostic and screening tests led to underestimation of STD prevalence in all three trials but especially in Mwanza. Adjusted prevalences of curable STD were higher in Mwanza than in Rakai and Masaka.