A procedure is developed for estimation of the cholinesterase activity in biological materials immediately on the plates for micro-thin-layer chromatography using a chamber of original construction. The procedure involved incubation of cholesterol esters, used as substrates and biological materials containing the enzyme, directly on the chromatographic plates in the chamber and the subsequent chromatography of the hydrolysis products. The enzymatic activity was calculated on the basis of the decrease in the substrate content per min per mg of protein. The procedure is simple, convenient; it allows to vary the incubation time and to estimate the enzymatic activity in minimal samples of the biological material using only small amounts of reagents.