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RPMI 1640, also known as RPMI medium or Roswell Park Memorial Institute Medium, is a growth medium used in cell culture. RPMI 1640 was developed by George E. Moore, Robert E. Gerner, and H. Addison Franklin in 1966 at Roswell Park Memorial Institute, from where it derives its name. A modification of McCoy′s 5A medium (or RPMI 1630), it was originally formulated to support culture and expansion of lymphoblastoid cells in suspension, but can also support a wide range of anchorage dependent mammalian cells. RPMI contains 19 essential and non-essential amino acids including glycine, 11 vitamins, 6 salts, and sodium bicarbonate.

It has traditionally been used for growth of human lymphocytes. This medium contains a great deal of phosphate and is formulated for use in a 5% carbon dioxide atmosphere. RPMI 1640 has traditionally been used for the serum-free expansion of human lymphoid cells. RPMI 1640 uses a bicarbonate buffering system and differs from most mammalian cell culture media in its typical pH 8 formulation. Properly supplemented with serum or an adequate serum replacement, RPMI 1640 allows the cultivation of many cell types, especially human T/B-lymphocytes, bone marrow cells, and hybridoma cells.