This chapter introduces the basic biology of CD133 (alias prominin-1), a cholesterol-binding pentaspan membrane glycoprotein that has gained enormous interest in the stem cell field. As a cell surface antigen, CD133 has become a prominent marker used for the prospective isolation and characterization of cells with stem cell properties in neural development and adult brain as well as in cancer. Beyond its application as a new biological tool for tissue engineering and regenerative therapy, CD133 has highlighted new aspects of the visual system because mutations in the PROM1 gene cause retinal pathologies. Here, we will summarize the essentials regarding the basic molecular and cellular biology of CD133 necessary for the proper evaluation of its detection in a given cell population under physiological or pathological conditions with a special focus on the neural system across species.