The incorporation of saturated fatty acids into the phospholipids of cultured mouse peritoneal macrophages was shown to lead to reduced endocytic activity (Mahoney, E. M., Hamill, A. L., Scott, W. A., and Cohn, Z. A. (1977)Pmc. Natl. Ad Sei. U. S. A. 74,4895-4899). More detailed analyses concerning the kinetics of fatty acid substitution, lipid composition, membrane bilayer structure, and the fatty acid enrichment of a plasma membrane-derived organelle have now been performed. In these studies, macrophages were cultivated in serumless medium containing 10 pg/ml of fatty acid complexed with bovine serum albumin. Under these conditions, the rate of incorporation of 14C-fatty acid into cell phospholipids was determined to be 8.8 nmol/ h/mg of cell protein during the first 8 to 10 h, after which incorporation into phospholipids ceased. Although the fatty acyl composition of phospholipids was substantially altered, the relative amounts of the phospholipids and the molar cholesterol/phospholipid ratio remained unchanged during this period. Phagolysosomes, derived largely from plasma membrane, were isolated from macrophages labeled with I4C-fatty acid and similar enrichment of this fraction in the activity of the plasma membrane marker 5’-nucleotidase (4.1fold) and in 14C radioactivity (3.7-fold) suggested that the fatty acid supplement was incorporated into plasma membrane. Spin label electron spin resonance studies indicated that the membrane lipid bilayer of macrophages enriched with saturated fatty acids was more rigid than that of control cells. The reversibility of fatty acid enrichment and the concomitant increase in endocytic activity were shown in washout experiments. The cultivation of 14C-fatty acid-enriched macrophages in serum-containing medium led to the removal of 65% of the radiolabeled fatty acid from cell phospholipids within 24 h and was accompanied by a 123% increase in pinocytic activity. These results suggested that an increased content of saturated fatty acid in plasma membrane phospholipids leads to both reduced bilayer fluidity and reduced ability of the cell to interiorize plasma membrane.