DnaA protein is the initiator of chromosomal DNA replication in Escherichia coli. We examined the influence of artificial mixed membrane composed of synthetic acidic (phosphate) lipid and basic (ammonium) lipid on the affinity of DnaA protein for ATP. Two sets of acidic and basic lipids with distinguishable numbers of hydrophobic alkyl chains were devised. Synthetic membranes made of the sole acidic lipid but not the basic bilayers inhibited the ATP binding to DnaA protein and stimulated the release of ATP from the ATP-DnaA complex. The basic bilayer-forming compounds served as the matrix for the guest acidic lipids. Acidic lipids dispersed in the basic matrix membrane had little effect on ATP binding and on ATP release. Conversely, acidic lipids forming cluster structures in the mixed artificial membranes inhibited the ATP binding and stimulated the release of ATP. These observations suggest that in mixed lipid bilayers, a cluster structure of acidic lipids seems to be an important parameter to decrease the affinity of DnaA protein for ATP.