Embryonic stem (ES) cells, the totipotent outgrowths of bias-tocysts^1,2, can be cultured and manipulated in vitro and then returned to the embryonic environment where they develop normally and can contribute to all cell lineages^3–9. Maintenance of the stem-cell phenotype in vitro requires the presence of a feeder layer of fibroblasts^1,2,10 or of a soluble factor, differentiation inhibitory activity (DIA) produced by a number of sources^5,11,12; in the absence of DIA the ES cells differentiate into a wide variety of cell types. We recently noted several similarities between partially purified DIA and a haemopoietic regulator, myeloid leukaemia inhibitory factor (LIF), a molecule which induces differentiation in Ml myeloid leukaemic cells and which we have recently purified, cloned and characterized^13–18. We demonstrate here that purified, recombinant LIF can substitute for DIA in the maintenance of totipotent ES cell lines that retain the potential to form chimaeric mice.