Intramammary infections of ruminant livestock impact animal health, welfare, and the economics of the dairy industry worldwide. A major hurdle to studying the ruminant mammary gland is the lack of in vitro tools to perform research on the mammary response to infection under laboratory conditions. Here, we propose to develop ruminant derived three-dimensional culture models (“organoids”) which represent the internal cellular structure of the mammary gland. Firstly, we will establish a tissue bank from non-lactating and lactating sheep and cow at different ages and various stages of the lactation cycle. Secondly, we will identify a panel of ruminant epithelial cell-type specific antibody markers using the tissue sections from lactating and non-lactating cows and sheep. Thirdly, we will use the panel of basal and luminal epithelium-specific antibody markers to define the structural and cellular organization in mammary epithelial organoids. We will induce lactation by hormonal stimulation of the organoids and characterize the response by gene expression and proteomic analysis. Finally, we will demonstrate the utility of ruminant mammary organoids as a model for intramammary bacterial infection. This will provide a well characterized laboratory tool in support of the development of new mastitis control measures.