Specular neutron reflectivity was used to study the time course and nature of the interaction of the positively charged, peripheral membrane protein cytochrome c with supported bilayers of zwitterionic 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) containing the anionic lipid 1-palmitoyl-2-oleoyl-glycero-3-phosphatidylserine (POPS). The supported bilayers were prepared by deposition on silicon blocks of two monolayers of DOPC, the second of which contained either 10 or 20 mol % POPS at surface pressures of either 15 or 20 mN/m using a combination of Langmuir-Blodgett and Schaefer deposition techniques. Each supported bilayer was initially characterized by specular neutron reflectivity using subphases of 10 mM NaCl aqueous solutions. Regardless of POPS content and bilayer deposition pressure, the molecular architecture of the bilayers; was similar. The addition of cytochrome c resulted in an almost immediate change in reflectivity, which was well modeled by assuming that an additional layer was present next to the outer leaflet of the bilayer. The thickness of this layer, which contained the volume fraction of similar to 15% protein, was similar to 30 angstrom (consistent with the cross-section of a single cytochrome c molecule). The addition of cytochrome c to the subphase also resulted in a change in the structure of the phospholipid bilayer, suggesting some penetration of cytochrome c into the bilayer. Specular neutron reflectivity studies after careful washing with solvent showed that although most of the protein was washed off by flushing 10 mM NaCl D2O through the cell a small amount remained both within the bilayer and bound to the membrane surface.