RPMI 1640 Medium was developed at Roswell Park Memorial Institute in 1966 by Moore and his co-workers. A modification of McCoy′s 5A Medium, it was formulated to support lymphoblastoid cells in suspension culture, but it has since been shown to support a wide variety of cells that are anchorage dependent. Originally intended to be used with a serum supplement, RPMI 1640 has been shown to support several cell lines in the absence of serum. It has also been widely used in fusion protocols and in the growth of hybrid cells.

RPMI-1640 Medium has been used to culture murine fibroblast cells. It has also been used to culture human ovarian and rat substantia nigra cells.