A fragment consisting of residues 584-1071 of the mouse Son-of-sevenless 1 (mSos1) protein was found to be sufficient for stimulation of the guanine nucleotide exchange of Ras in vitro, which defines the CDC25 homology (CDC25H) domain of mSos1. Furthermore, we found that the CDC25H-domain fragment activated the extracellular signal-regulated protein kinases (ERKs), and was mainly membrane localized, when expressed in unstimulated human embryonic kidney 293 cells. Then, we examined the roles of other mSos1 domains in autoinhibition of the CDC25H-domain functions in unstimulated cellular environments. First, longer fragments that have the CDC25H domain and the following proline-rich Grb2-binding domain exhibited negligible membrane localization, and accordingly much lower ERK-activation activities, under serum-starved conditions. On the other hand, the preceding Pleckstrin-homology (PH) domain affects neither the ERK-activation activity nor the membrane-localization activity of the CDC25H domain. By contrast, the cells expressing a fragment containing the Dbl homology (DH) domain in addition to the PH and CDC25H domains exhibited remarkably low ERK activities under serum-starved conditions. This autoinhibitory effect of the DH domain on the CDC25H-domain function was shown to be relieved when cells were stimulated with epidermal growth factor. The DH-domain extension affected neither the in vitro guanine nucleotide exchange activity nor the membrane-localization activity of the CDC25H domain. Therefore, one of the roles of the DH domain is to exert an autoinhibition over the CDC25H-domain function on the cell membrane, in the absence, but not in the presence, of extracellular stimuli.