It has been established that the hydrolysis of EGTA-acetoxymethylester (AME) by red blood cells is about the tenth of the hydrolysis of acetylthiocholine (Ac-S-Ch). This splitting of AME could be inhibited by about 50% by prostigmine at a concentration of 0.75 X 10(-5) mol/l, while the splitting of Ac-S-Ch was totally inhibited by the same prostigmine concentration. The hydrolysis of AME by the so-called white-ghost preparation was considerably inhibited by prostigmine (KI = 5 X 10(-8) mol/l), and this inhibition proved to be a competitive one. The splitting of AME by membrane-free cytosol fraction could not be inhibited by prostigmine. Human red blood cells do not hydrolyse EGTA-ethylester (EE). This compound decomposes spontaneously at room-temperature, its reaction-product formed in the Hestrin-reaction is unstable, the developed colour gradually turns pale. On the other hand, AME does not hydrolyse spontaneously at room-temperature and the colour-intensity of its Hestrin-reaction does not decrease with time. Using chelator-and dye-indicator esters to reach different concentrations of free chelators and dye-indicators intracellulary (IC), the extracellular hydrolysis of esters has to be taken into account or this external breakdown has to be inhibited.