Mast cells are cells of the innate immune system whose biological responses are markedly modulated by effector molecules of adaptive immunity, i.e., antibodies. They thus contribute to anti-infectious defense but also to antibody-dependent inflammatory responses. They are especially well known as inducers of allergic reactions. They are widely distributed in most tissues, but in low numbers. They are not readily purified, and with a poor yield. For these reasons, means to generate large numbers of homogenous non-transformed mast cells have been developed. We describe here (1) fractionation methods suitable for purifying mouse or rat peritoneal mast cells and for purifying human mast cells of various origins, and (2) conditions for generating pure cultured mast cell populations from mouse, rat, and human tissues.