The substrate requirements for the catalytic activity of the mouse Cdc25 homolog Guanine nucleotide Release Factor, GRF, were determined using the catalytic domain of GRF expressed in insect cells and E. coli expressed H-Ras mutants. We found a requirement for the loop 7 residues in Ras (amino acids 102 to 105) for stimulation of guanine nucleotide release. The dependence on the switch I and II regions of Rasp21 (encompassing the residues that shift position in the GTP- versus GDP-bound protein), which had been seen with Sdc25-mediated exchange, was also found for GRF. In addition, the sensitivity of H-Ras to GRF was abolished when residues 130-139 were replaced by proline-aspartic acid-glutamine, whereas substitution of the entire loop 8 (residues 123-130 replaced by leucine-isoleucine-arginine) had no effect on the stimulation of guanine nucleotide release by GRF. Substrate activity of Ras proteins were independent of their post-translational processing, GDP release was stimulated threefold more effectively by GRF than was GTP release, and no major differences were found between the mammalian N-, H- and K-Ras proteins. Examining the responsiveness of the Ras protein from S. pombe and the human Ras like proteins RhoA, Rap1A, Rac1 and G25K revealed a strict Ras specificity; of these only S. pombe Ras was GRF sensitive.