The adsorption of membrane-associated protein cytochrome c to anionic lipid bilayers of dioleoyl phosphatidylglycerol was studied in low ionic strength physiological buffer using atomic force microscopy. The bilayers were supported on polylysinated mica. The formation of stable, single lipid bilayers was confirmed by imaging and force spectroscopy. Upon addition of low concentrations of cytochrome c, protein molecules were not topographically visible on the lipid bilayer-buffer interface. However, the forces required to punch through the bilayer by indentation using the atomic force microscopy probe were significantly lower after protein adsorption, which suggest that the protein inserts into the bilayer. Moreover, the apparent thickness of the bilayer remained unchanged after cytochrome c adsorption. Yet, mass spectroscopy and visible light absorption spectroscopy confirmed the presence of cytochrome c in the lipid bilayers. These results suggest that 1), cytochrome c inserts into the bilayer and resides in its hydrophobic core; 2), cytochrome c insertion changes the mechanical properties of the bilayer significantly; and 3), bilayer force spectroscopy may be a useful tool in investigating lipid-protein interactions.