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Amphipols (APols) are amphipathic polymers with the ability to substitute detergents to keep membrane proteins (MPs) soluble and functional in aqueous solutions. APols also protect MPs against denaturation. Here, we have examined the ability of APol-trapped MPs to be analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). For that purpose, we have used ionic and nonionic APols and as model proteins (i) the transmembrane domain of Escherichia coli outer membrane protein A, a β-barrel, eubacterial MP, (ii) Halobacterium salinarum bacteriorhodopsin, an α-helical archaebacterial MP with a single cofactor, and (iii, iv) two eukaryotic MP complexes comprising multiple subunits and many cofactors, cytochrome b(6)f from the chloroplast of the green alga Chlamydomonas reinhardtii and cytochrome bc(1) from beef heart mitochondria. We show that these MP/APol complexes can be readily analyzed by MALDI-TOF-MS; most of the subunits and some lipids and cofactors were identified. APols alone, even ionic ones, had no deleterious effects on MS signals and were not detected in mass spectra. Thus, the combination of MP stabilization by APols and MS analyses provides an interesting new approach to investigating supramolecular interactions in biological membranes.