Abstract A progressive evolution of sensitivity to TTX and to Mn 2+ was observed in isolated chick embryo heart cells in culture. TTX-insensitivity progressively develops and affects the whole cell population after 48 h of cultivation. At the same time, spontaneously beating cells become sensitive to Mn ions which inhibit their activity. Insulin prevents the decrease of TTX-sensitivity of the cell population during the 4 to 48 h time of cultivation. It is assumed that the artificial conditions of the tissue culture method are partly responsible for the changes of the cell membrane properties. An improvement of these conditions by adding insulin to the culture medium can inhibit the loss of normal properties such as TTX-sensitivity. Whether insulin acts by influencing the value of the resting potential or by controlling protein synthesis in the cultured cells remain unknown.