In a glycerol auxotroph of Staphylococcus aureus, the deprivation of glycerol affected the formation of certain membrane components. (i) There was synthesis of fatty acids at the predeprivation rate even though the fatty acids synthesized accumulated as free fatty acids rather than as esterified fatty acids; (ii) there was a complete cessation of phospholipid and vitamin K isoprenologue biosynthesis; (iii) there was conservation of the glycerol esters of the complex phospholipids and glucolipids; (iv) there was an immediate decrease in the rate of synthesis of monoglucoslydiglyceride (30%) and diglucosyldiglyceride (60%); (v) there was a 50% decrease in the rate of synthesis of the polar and nonpolar carotenoids; (vi) there was synthesis of protoheme, heme a, and nonspecific membrane protein at the predeprivation rate; and (vii) there was an abrupt cessation in the formation of new, functional glycine transport activity.