Detergents are customarily used to solubilize cell membranes and keep membrane proteins soluble in aqueous buffers, but they often lead to irreversible protein inactivation. Hemifluorinated amphiphiles with hybrid hydrophobic chains have been specifically designed to minimize the denaturating propensity of surfactants toward membrane proteins. We have studied the physical-chemical and biochemical properties of lactobionamide surfactants bearing either a hydrogenated, a fluorinated or a hemifluorinated chain (respectively H-, F-, and HF-Lac). We show that the dual composition of the hydrophobic chain of HF-Lac endows it with unusual physical-chemical properties as regards its critical micellar concentration, interfacial area per molecule, and behavior upon reverse phase chromatography. Analytical ultracentrifugation shows that, whereas H-Lac assembles into well-defined micelles, F-Lac and HF-Lac form large and heterogeneous assemblies, whose size increases with surfactant concentration. Molecular dynamics calculations suggest that F-Lac forms cylindrical micelles. The ability of HF-Lac to keep membrane proteins soluble was examined using the cytochrome b(6) f complex from Chlamydomonas reinhardtii's chloroplast as a model protein. HF-Lac/b(6) f complexes form particles relatively homogeneous in size, in which the b(6) f complex is as stable or markedly more stable, depending on the surfactant concentration, than it is in equivalent concentrations of hydrogenated surfactants, including H-Lac.