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Background Globally, women have higher herpes simplex virus type 2 (HSV-2) prevalence than men; data from observational studies suggest a possible association of HSV-2 acquisition with use of intramuscular depot medroxyprogesterone acetate (DMPA-IM). Methods Within a randomized trial of the effect of 3 contraceptive methods—DMPA-IM, a copper intrauterine device (IUD), and a levonorgestrel (LNG) implant—on human immunodeficiency virus (HIV) acquisition, we assessed HSV-2 acquisition. HSV-2 and HIV seronegative women, aged 16–35 years, and seeking effective contraception were followed for 12–18 months at 12 sites in Eswatini, Kenya, South Africa, and Zambia from 2015 to 2018. HSV-2 serologic testing was done at enrollment and final study visits. Intention-to-treat analysis using Poisson regression with robust standard errors compared HSV-2 incidence by contraceptive method. Results At baseline, 4062 randomized women were HSV-2 seronegative, of whom 3898 (96.0%) had a conclusive HSV-2 result at their final study visit. Of these, 614 (15.8%) acquired HSV-2, at an incidence of 12.4/100 person-years (p-y): 10.9/100 p-y among women assigned DMPA-IM, 13.7/100 p-y the copper IUD, and 12.7/100 p-y the LNG implant. Incidence rate ratios (IRR) for HSV-2 acquisition were 0.80 (95% confidence interval [CI],.65–.97) for DMPA-IM compared with copper IUD, 0.86 (95% CI,.71–1.05) for DMPA-IM compared with LNG implant, and 1.08 (95% CI,.89–1.30) for copper IUD compared with LNG implant. HSV-2 acquisition risk was significantly increased among women who also acquired HIV during follow-up (IRR 3.55; 95% CI, 2.78–4.48). Conclusions In a randomized trial, we found no association between HSV-2 acquisition and use of 3 contraceptive methods. Trial registration ClinicalTrials.gov number NCT02550067. [ABSTRACT FROM AUTHOR]