Background: HIV prevalence and incidence among sexually active women in peri-urban areas of Ladysmith, Edendale, andPinetown, KwaZulu-Natal, South Africa, were assessed between October 2007 and February 2010 in preparation for vaginalmicrobicide trials.Methodology/Principal Findings: Sexually active women 18–35 years, not known to be HIV-positive or pregnant weretested cross-sectionally to determine HIV and pregnancy prevalence (798 in Ladysmith, 1,084 in Edendale, and 891 inPinetown). Out of these, approximately 300 confirmed non-pregnant, HIV-negative women were subsequently enrolled ateach clinical research center (CRC) in a 12-month cohort study with quarterly study visits. Women in the cohort studies wererequired to use a condom plus a hormonal contraceptive method. HIV prevalence rates in the baseline cross-sectionalsurveys were high: 42% in Ladysmith, 46% in Edendale and 41% in Pinetown. Around 90% of study participants at each CRCreported one sex partner in the last 3 months, but only 14–30% stated that they were sure that none of their sex partnerswere HIV-positive. HIV incidence rates based on seroconversions over 12 months were 14.8/100 person-years (PY) (95% CI9.7, 19.8) in Ladysmith, 6.3/100 PY (95% CI 3.2, 9.4) in Edendale, and 7.2/100 PY (95% CI 3.7, 10.7) in Pinetown. The 12-monthpregnancy incidence rates (in the context of high reported contraceptive use) were: 5.7/100 PY (95% CI 2.6, 8.7) inLadysmith, 3.1/100 PY (95% CI 0.9, 5.2) in Edendale and 6.3/100 PY (95% CI 3.0, 9.6) in Pinetown.Conclusions/Significance: HIV prevalence and incidence remain high in peri-urban areas of KwaZulu-Natal.