Addition of sonicated dispersions of cholesterol to peptone-salt-vitamin medium resulted in the metabolism of the sterol byHartmanella culbertsoni. Trophozoite multiplication was stimulated at 1–5 mg/litre, but retarded at 10–20 mg/litre. When cholesterol was added to the medium, incorporation of [1,2-14C] -acetate into neutral lipid, phospholipid, non-saponifiable and cholesterol fractions of the amoebae was significantly reduced. Cholesterol ester was detected in the medium but phospholipids were not released. Addition of cholesterol stimulated the activity of lysosomal acid phosphatase, acid deoxyribonuclease and cathepsin B but did not affect 5′-nucleotidase, adenosine triphosphatase, alkaline phosphatase, glucose-6-phosphatase, succinate dehydrogenase and cytochrome C oxidase.