In Ghana, breastfeeding is the most economically feasible and culturally acceptable method of infant feeding. HIV positive mothers are advised to exclusively breastfeed for 6 months and to stop as soon as it is feasible and safe. Knowing when to expect milk production to increase is important especially in counseling HIV positive women who intend to exclusively breastfeed, to prevent the introduction of other foods which would affect exclusive breastfeeding (EBF) duration. ^ This was a longitudinal cohort study, with quantitative and qualitative components, investigating the association between maternal HIV status and the reported onset of lactation (OL), specifically: (1) to validate the reported maternal perception of the OL based on longitudinal infant weight measurements and test weights of infant breastmilk intakes, (2) to determine the influence of HIV on the OL, (3) to assess the influence of HIV status on breast health during the first month postpartum, (4) to determine the influence of HIV on EBF during the first 3 months postpartum and (5) to determine the barriers to as well as the benefits and predictors of EBF. ^ Participants were 425 mothers participating in the Research to Improve Infant Nutrition and Growth (RIING) project recruited from three antenatal clinics in the Eastern region of Ghana, based on HIV status. OL was assessed by maternal perception which was validated using sub-samples for infant test weight measurements (n = 40) and daily infant weight measurements for 2 weeks postpartum (n = 150). Multivariate logistic regression was used to identify predictors of very early onset of lactation (VEOL; OL \u3c 6 hours). Predictors of VEOL included: HIV-status (OR=2.68; p=0.014), multiparity (OR=2.93; p=0.009), vaginal delivery (OR=2.55; p=0.035) and having a male child (OR=1.86; p=0.032). ^ The qualitative study shows widespread EBF knowledge in this population with antenatal clinics being the major source of infant feeding information. The health and well-being of the infant were identified as the main motivation for EBF, while perceived breastmilk unavailability, employment and pressure from family were the major barriers to EBF. ^ Our findings indicate an association between maternal HIV status and VEOL.