The Soret absorption band has been utilized as a probe for the adsorption of cytochrome c to the surface of a fused silica prism in direct contact with bulk protein solutions of various concentrations and pH. Employing linear polarized light and a single-pass total internal reflection absorption technique, we examined in detail the adsorption isotherm, molecular orientation, packing density, and conformational change of the protein bound to the bare (hydrophilic) and silanized (hydrophobic) glass surfaces. An adsorbate density of Gamma = 1.4 x 10(13) molecules/cm(2) was determined for the hydrophilic substrate at pH 7.2 and C-b = 11 muM, indicating that the protein molecules are essentially closely packed on the surface at saturation. The packing density is sensitive to the solution pH as well as the surface hydrophobicity, a result that the protein-surface interaction is governed by both electrostatic and hydrophobic forces. The same forces also govern the molecular orientation, yielding an angle of theta(mu), = 41degrees between the heme plane and the surface normal at neutral pH. The angle is retained over a wide pH range (4-9) and is fairly independent of the surface coverage on both the hydrophilic and hydrophobic substrates. Reorientation of the protein occurs (41degrees --> 20degrees) at pH approximate to 3, when the cyt c unfolds and the hydrophobic force becomes dominant in the adsorption process.