Techniques (push-pull cannula, cup, brain ventricles perfusion) allowing estimation of the amount of the ACh released in vivo from the brain are described. The main attention is paid to biochemical, physiological and morphological factors influencing the amount of ACh released and available for estimation. Conditions of experiments for each of these techniques are described in details. The amount of ACh released in different physiological states of several animal species is compared. Typical applications of all three methods are given.