Calcium ion is required for ACTH to exert a lipolytic action on rat epididymal adipose tissue slices. Other metal ions such as Mg++, Zn++, Cu++, Ba++ and Mn++ can not substitute for Ca++. In the adipose tissue homogenate the action of ACTH disappears and calcium ion stimulates lipolysis. Besides, ACTH increases the incorporation of calcium ion into the adipose tissue slices. All these results suggest that the lipolytic effect of ACTH might be mediated through calcium ion.