Palladium di (sodium alizarinmonosulfonate) is a highly efficient catalyst for the hydrogenation of unsaturated fatty acids esterified in lipids of model or biological membranes, enabling the study of the relationship between function and the physical state of membranes. However, the catalyst shows a complex behavior in the action of molecular hydrogen and oxygen, giving rise to the formation of at least four products. Two of these are free radicals. Owing to this complexity, precise control of the reaction requires pretreatment of the catalyst. When partial hydrogenation of the palladium complex is followed by air oxidation, a catalyst solution is produced which is stable on air and maintains catalytic hydrogenation activity for several days. This form of the catalyst induces hydrogenation of unsaturated lipids with no induction period making a strict timing of the procedure possible. Of the several other factors affecting the outcome of membrane hydrogenations, one of the most important is the accessibility to the catalyst of particular membrane regions or lipid pools. Differences in accessibility may arise as a consequence of different local microviscosities or their change during hydrogenation, of the appearance of distinct liquid crystalline phases, and of strong protein-lipid interactions. Obviously, in case of whole-cell hydrogenations, the accessibility is influenced by the spatial separation of the organelles, as well.