The effect of fat absorption upon the phospholipid pool size of the intestinal mucosal cells was determined in rats receiving fatty emulsions as a bolus by stomach tube or as multiple meals in the form of fat-laden laboratory chow. The phospholipid content of the mucosal scrapings and of the isolated villus cells was determined 3 to 34 h after the meals and was compared with the phospholipid content of cells from similar animals receiving water alone or 10% sucrose in water. It was shown that continuously fed animals averaged 5-10% and single meal fed animals up to 40% higher phospholipid content in their mucosal cells than the corresponding controls, when compared per milligram cell protein. The expansion of the phospholipid pool involved all phospholipid classes and correlated well with the phospholipid composition of prechylomicrons and of microsomal membranes, which undergo a significant proliferation during fat absorption. The apparent lower expansion of the phospholipid pool in the continuously fed animals correlated with the lower triacylglycerol content of the lumen and of the cells at these times.