Protein translocation in Escherichia coli is mediated by the SecA ATPase bound to the SecYEGmembrane protein complex. SecA translocation ATPase activity as well as protein translocation isdependent on the presence of negatively charged lipids. By using a phospholipid with an acyl chainlinked photoactivatable group, the lipid accessibility of SecA bound at the translocase was explored. SecAbound to lipid vesicles containing negatively charged lipids was found to be readily accessible for labelingby the photoactivatable phospholipid. The presence of an excess amount of SecYEG complex resultedin a remarkable reduction in the amount of lipid-accessible SecA irrespective of the nucleotide-boundform of SecA. These data demonstrate that the SecYEG-bound SecA is largely shielded from thephospholipid acyl chains and suggest the presence of two distinct pools of membrane-bound SecA thatdiffer in the degree of lipid association.