The vital staining mechanism of alizarin red S (ARS) was investigated. The total calcium content of rat blood was determined after intraperitoneal and intracardial administration of acidic, neutralized and calcium-supplemented ARS. The administration of ARS causes, irrespective of chemical state, a remarkable decrease in blood volume and a significant decrease in the serum/blood ratio. The total calcium content of rat serum was noticeably increased, more in the case of intracardial than in the case of intraperitoneal administration. The calcium values determined in this work were the sums of free and bound calcium. The increase in calcium content even in those cases where ARS was administered without calcium indicates a physiological ability to keep the concentration of free calcium constant in the blood. The highest calcium values were found when calcium-supplemented ARS was administrated. The physiological activity, however, seems to be higher with acidic ARS, which also contributed to the vital staining effect on rat skull. The best staining with least inconvenience was achieved using neutralized ARS for in vivo staining.