SecA is found in the cytosol and bound to the plasma membrane of Escherichia coli. Binding occurs either with high affinity at SecYEG or with low affinity to lipid. Domains of 65 and 30 kDa of SecYEG-bound SecA insert into the membrane upon interaction with preprotein and ATP. Azide blocks preprotein translocation, in vivo and in vitro, through interacting with SecA and preventing SecA deinsertion. This provides a measure of the translocation relevance of each form of SecA membrane association. We now report that azide acts exclusively on SecA that is cycling at SecYEG and has no effect on SecA lipid associations. SecA molecules recovered with sucrose gradient-purified inner membrane vesicles ('endogenous' SecA) support translocation at the same rate as 'added' SecA molecules bound at SecYEG. Both endogenous and added SecA yield the same proteolytic fragments, which are distinct from those obtained from SecA once it has inserted into membranes at SecYEG or from SecA at lipidic sites. Endogenous and added SecA differ, however, in their resistance to urea extraction. The translocation supported by either endogenous or added SecA is blocked by azide or by antibody to SecY. We conclude that SecA functions in preprotein translocation only through cycling at SecYEG.