SecA-lipid interactions are believed to be important for the translocation of precursor proteins across the inner membrane of Escherichia coli [Lill, R., Dowhan, W., & Wickner, W. (1990) Cell 60, 271-280]. SecA insertion into the phospholipid bilayer could a role in this process. We investigated this possibility by studying the interactions between SecA and different phospholipids using the monolayer technique. It was established that SecA is surface-active and can insert into lipid monolayers. This insertion was greatly enhanced by the negatively charged lipids DOPG and Escherichia coli cardiolipin. Insertion of SecA into these negatively charged lipids could be detected up to initial surface pressures of 34 mN/m for DOPG and 36 mN/m for Escherichia coli cardiolipin, implying a possible role for negatively charged lipids in the insertion of SecA in biological membranes. High salt concentrations did not inhibit the SecA insertion into DOPG monolayers, suggesting not only an electrostatic but also a hydrophobic interaction of SecA with the lipid monolayer. ATP decreased both the insertion (factor 2) and binding (factor 3) of SecA to DOPG monolayers. ADP and phosphate gave a decrease in the SecA insertion to the same extent as ATP, but the binding of SecA was only slightly reduced. AMP-PNP and ATP-gamma-S did not have large effects on the insertion or on the binding of SecA to DOPG monolayers. The physiological significance of these results in protein translocation is discussed.