Freshly isolated hairy cells from the peripheral blood of patients with hairy cell leukemia (HCL) synthesize 3–5-fold greater amounts of cholesterol, lanosterol, and squalene from [1–14C]-acetate than do normal human peripheral blood mononuclear cells under basal conditions of culture (i.e., in the presence of low-density lipoprotein). HCL cells also exhibit an eightfold increase in the activity of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase. These changes cannot be ascribed to increased rates of cellular proliferation in the HCL cells, nor are they a consequence of an increased rate of loss of newly synthesized cholesterol into the culture medium. The increased rate of cholesterol biosynthesis in HCL cells may result in an increase in their total cellular cholesterol content, as well as in an increase in their plasma membrane cholesterol:phospholipid molar ratio. These changes, in turn, are probably responsible for some of the clinical manifestations of this disease.