Breastfeeding (BF) women are an important population for biomedicalHIV prevention strategies, but they are rarely included in trials. The 25-mg dapivirinevaginal ring (VR) reduced women’s risk of sexually transmitted HIV infection in twophase 3 trials conducted in Africa. We conducted a phase 1, open-label study (MTN-029/IPM 039) of dapivirine VR use among lactating women in Pittsburgh, PA, andBirmingham, AL, USA. MTN-029/IPM 039 enrolled 16 healthy adult women who hadalready weaned their infants but were still able to express breast milk. Women wereinstructed to use the VR continuously for 14 days and provided milk, plasma, andcervicovaginal fluid (CVF) samples for pharmacological analysis. No infants were exposedto the drug, but infant dosage was estimated according to FDA guidance. Adverseevents (AEs) were collected at all contacts. The study was completed with100% participant retention. Median dapivirine concentrations were 676 pg/ml inbreast milk, 327 pg/ml in plasma (milk/plasma ratio ?2.0), and 36.25 ng/mg in CVF.Six participants experienced 10 total AEs, none of which required VR discontinuation.The estimated mean daily infant dosage was 74.3 ng/kg/day. In this first studyof dapivirine exposure during lactation, dapivirine VR use was associated with lowerconcentrations of detectable dapivirine in milk and plasma than in CVF samples anda favorable safety profile. Estimated daily levels of infant dapivirine exposure werealso low. Additional studies are needed to evaluate longer periods of dapivirine VRuse among BF mother-infant pairs living in regions with higher incidence of sexuallytransmitted HIV infection. (This study has been registered at ClinicalTrials.gov underregistration no. NCT02808949.)