The mechanism underlying the interaction of the Escherichia coli signal recognition particle (SRP) receptor FtsY with the cytoplasmic membrane is not fully understood. We investigated this issue by utilizing active (NG+1) and inactive (NG) mutants of FtsY. In solution, the mutants comparably bind and hydrolyze nucleotides and associate with SRP. In contrast, a major difference was observed in the cellular distribution of NG and NG+1. Unlike NG+1, which distributes almost as the wild-type receptor, the inactive NG mutant accumulates on the membrane, together with ribosomes and SRP. The results suggest that NG function is compromised only at a later stage of the targeting pathway and that despite their identical behavior in solution, the membrane-bound NG-SRP complex is less active than NG+1-SRP. This notion is strongly supported by the observation that lipids stimulate the GTPase activity of NG+1-SRP, whereas no stimulation is observed with NG-SRP. In conclusion, we propose that the SRP receptor has two distinct and separable roles in (i) mediating membrane targeting and docking of ribosomes and (ii) promoting their productive release from the docking site.