Concentrations of HIV-1 RNA and DNA in mucosal compartments influence the risk of sexual transmission and mother-to-child transmission of HIV-1. Breast milk production is physiologically regulated such that supply is a function of infant demand, but whether demand also influences HIV-1 dynamics in breast milk is unknown. We tested whether minor and major changes in feeding frequency influence breast milk viral concentrations in 958 HIV-1-infected women and their infants followed, for 24 months during a trial in Lusaka, Zambia. Women were randomized to wean abruptly at 4 months or to continue breast-feeding for a duration of their own choosing. Two weeks after breast-feeding cessation (4.5 months), HIV-1 concentrations in breast milk were substantially higher (median RNA, 2708 copies/ml; DNA, 14 copies/ml) than if breast-feeding continued (median RNA, <50 copies/ml; DNA, <1 copy/ml; P < 0.0001). Among those continuing breast-feeding, HIV-1 concentrations in milk were higher if breast-feeding was nonexclusive (median RNA, 293 copies/ml; DNA, 2 copies/ml; P = 0.0006). Elevated milk viral concentrations after stopping breast-feeding explained higher than expected rates of late postnatal HIV transmission in those who weaned early. Changes in the frequency of breast-feeding peri-weaning and with nonexclusive breast-feeding influenced milk viral concentrations. This may explain the reduced risk of HIV-1 transmission associated with exclusive breast-feeding and why early weaning does not achieve the magnitude of HIV prevention predicted by models. Our results support continuation of maternal antiretroviral drug interventions over the full duration of time when any breast milk exposures may occur after planned weaning.