Metanephric organ culture, or ex vivo embryonic kidney culture, was developed in the mid-twentieth century as a means to understand the development of the mammalian kidney and was used in early studies of polycystic kidney disease to explore mechanisms of renal cyst initiation by non-genetic factors. Following the identification of cystogenic genes, a resurgence of the use of metanephric organ culture occurred and has yielded insight into basic mechanisms of cystic dilation; facilitated identification of pathogenic pathways and potential therapeutic targets; and provided a system for evaluating therapeutic agents. This chapter provides detailed, step-by-step protocols with rationale and tips for the establishment, maintenance and treatment of metanephric organ cultures, and for performance of the most commonly employed secondary analyses of these cultures.