In this study, we have established that FtsY, the E. coli homolog of the mammalian signal recognition particle (SRP) receptor, is a GTP-binding protein which displays intrinsic GTPase activity. GTP was found to influence the protease sensitivity of FtsY indicative of a conformational change. FtsY mutated in the 4th GTP-binding consensus element displayed reduced GTP-binding and -hydrolysis which correlated with a reduced ability to interact with SRP. Overexpression of the mutant proteins had a stronger inhibitory effect on protein translocation than overexpression of wild-type FtsY. These observations suggest that in E. coli GTP is important for proper functioning of FtsY in protein-targeting.