The bacterial translocon interacts with both SecA-bound preproteins and nascent chain-ribosome complexes during Sec and signal recognition particle-dependent protein translocation, respectively. In their inactive state, translocons are saturated with ribosomes and SecA protein, reflecting the inherent affinity of these components for one another. We found that SecA and ribosomes are bound simultaneously and noncompetitively to a common set of inactive translocons. Furthermore, we demonstrate that at a later stage in binding, SecA possesses a ribosome-translocon dissociation activity that is coupled to its ATP-dependent membrane insertion and retraction cycle that drives protein translocation. This novel activity is presumably important in the commitment of the translocon to the Sec-dependent pathway. These results also provide a rationale for the compatibility and regulation of multiple protein translocation pathways that each makes distinct demands on a common translocon core.