Abstract: The ATP synthase and complex I in mitochondria are membrane bound multisubunit assemblies of both hydrophilic and hydrophobic proteins. Hitherto, the mass spectrometric measurement of their molecular masses has required that many of the hydrophobic proteins be analyzed separately from the other components in two separate experiments. Here, we describe a procedure that allows the molecular masses of all, or almost all, of the subunits of each complex to be measured in a single experiment. The key feature is a mobile phase, in which hydrophilic and hydrophobic components remain soluble, that is compatible with reverse phase chromatography. In this way, the masses of all 17 subunits of bovine ATP synthase, 14 of the 17 subunits of the enzyme from Saccharomyces cerevisiae, 42 of the 45 subunits of bovine complex I and all 28 of the subunits of bovine subcomplex Ialpha were measured. The method has been used to characterize the subunits of ATP synthases and complexes I from a variety of species, and for following the progress of mild trypsinolysis of ATP synthase. It could be applied to other respiratory and photosynthetic complexes, and in general to any protein complex that contains both hydrophilic and hydrophobic subunits.