The minimal active domain of the mouse CDC25 M m , a GDP/GTP exchange factor (GEF) active on H-ras protein, was determined by constructing several deletion mutants of the C-terminal domain of the protein. The functional activity of these fragments was analyzed for the ability to complement the yeast temperature sensitive mutation cdc25-1 and to catalyze the GDP/GTP exchange on Ras proteins in vitro. A C-terminal domain of 256 residues (CDC25 M m 1 0 0 5 - 1 2 6 0 ) was sufficient for full biological activity in vivo. Deletion of 27 C-terminal amino acids (CDC25 M m 1 0 0 5 - 1 2 3 3 ) abolished the complementing activity while deletion of 25 N-terminal residues (CDC25 M m 1 0 3 0 - 1 2 6 0 corresponding to the most conserved domain) led to a complete loss of expression. The results in vivo were supported by experiments in vitro. Highly purified CDC25 M m 1 0 0 5 - 1 2 6 0 , expressed in E. coli using the pMAL system, enhanced the GDP release from both H-ras p21 and S. cerevisiae Ras2p and its activity was nearly as high as that of CDC25 M m 9 7 4 - 1 2 6 0 . Comparison with the Cdc25p protein yielded further evidence that the minimal active domain of CDC25 M m is shorter than the yeast one.