PROJECT SUMMARY Antibiotic use is common among women of reproductive potential, including lactating women. However, little is known about the transmission and pharmacokinetics of specific antibiotics in human milk, nor the effects of antibiotic treatment on milk composition and quality including nutritional components, oligosaccharide profile, and the microbiome. Establishment of the infant gut microbiome is dependent on a dynamic maternal–infant microbiota exchange during early life, which is largely facilitated via breastfeeding. It is thus critical to better understand the impact of specific antibiotics on milk composition. Furthermore, it is essential to determine how maternal antibiotic use may impact infant outcomes, including growth, development, and immune response. Building on the infrastructure of the University of California San Diego Human Milk Research Biorepository, a highly qualified, multidisciplinary research team will prospectively enroll and interview 480 maternal/infant pairs at baseline, collect milk, skin, and stool samples, and monitor ongoing breastfeeding practices and maternal use of one of four commonly used antibiotics over time. From these, we will subsequently identify 40 maternal/infant pairs with maternal uptake of target antibiotic treatment, 40 matched maternal/infant pairs without antibiotic exposure, and 20 non-lactating maternal/infant pairs with maternal uptake of a target antibiotic treatment. From these 100 pairs, we will collect additional home and maternal and infant biomarkers of exposure at 3 timepoints corresponding to antibiotic initiation, mid-course, and end-course, collect interview and medical record data, and obtain a questionnaire assessment of infant development at 12 months post-partum. Data will be used to determine the amount and pharmacokinetics of target antibiotics in breast milk samples and to test the central hypotheses that (1) milk composition, oligosaccharide, and microbiome profiles will differ between groups by antibiotic treatment status; (2) microbiome and metabolomic measures will differ in treated and untreated breastfeeding groups; and (3) antibiotics will be detectable in the stool, skin, and home environment swabs of mothers who are treated with antibiotics but are not breastfeeding. We will also explore the impact on maternal antibiotic use on infant growth and development. Findings of this study will fill a critical gap in knowledge regarding the presence of commonly used antibiotics in breast milk and the effects of antibiotic treatment on both breast milk composition and quality as well as short- and long-term infant outcomes, including growth, development, gut health, and immune response. This will inform evidence-based development of specific guidance on antibiotic use during lactation and help support advice provided by healthcare providers to breastfeeding women.