Different oxidants regulate the staining ability, the hue, and the staining target in bone, cartilage and connective tissue in vitro in connection with alizarin red S (ARS). The use of oxidants should promote the staining ability on guinea pig bone, also slightly on rabbit bone, but not on rat bone. Quite selective staining of bone, cartilage and connective tissue was achieved in this study via ARS oxidation. In addition to oxidizing, complex formation, especially when regulating the staining target, may be considerable. In these twelve experiments with oxidizing agents, the most promising results as concerns selectivity were obtained with phosphomolybdic acid, ferric chloride, stannic chloride and chromium trioxide. As agents activating the staining ability, potassium persulfate seems best. Based on the oxidizing influence of potassium persulfate and according to the potentiometric and precipitation analyses, the coordinative bonding of Ca2+ to the ARS molecule seems obvious. When the β-phenolic group of ARS is partly oxidized and, on the other hand, partly ionized, indicating the titration range, Ca2+ should be bound to it. Oxidized α-quinonic oxygen begins to form a coordinative bond with the same Ca2+ and thus the chelate is formed. On the slightly alkaline side, where the α-phenolic group has its ionization ranges, the oxidant causes a clear broadening and increase in the complex formation effect, which seems to be an indication of reactions of the calcium ions with two oxidized and partly ionized ARS molecules.