Mycoplasma offer several unique advantages for investigating the mechanism controlling transfer and uptake of exogenous cholesterol and phospholipids by biomembranes, as their plasma membrane interacts directly with exogenous lipid donors and their endogenous lipid synthesis is restricted. Growing cells of five species of Mycoplasma were found to take up significant quantities of phosphatidylcholine and sphingomyelin as well as free and esterified cholesterol. In contrast, growing cells of three species of Acholeplasma failed to take up any of the exogenous phospholipids and incorporated only low amounts of free cholesterol and no esterified cholesterol. It is hypothesized that Mycoplasma species have receptors for serum lipoproteins and phospholipid-cholesterol vesicles that facilitate the transfer of cholesterol and phospholipids to the growing cell membrane. Our finding that gentle trypsin treatment of growing Mycoplasma capricolum cells decreased their cholesterol uptake ability by about 50% but did not affect cholesterol uptake by growing Acholeplasma laidlawii cells appears to support the existence of protein receptors for lipoproteins on the surface of Mycoplasma but not on Acholeplasma species. Digestion of membrane phospholipids by phospholipase A2 decreased the cholesterol-binding capacity of isolated A. laidlawii and M. capricolum membranes, roughly in proportion to the amount of phospholipid digested. The total removal of phosphatidylglycerol and diphosphatidylglycerol from A. laidlawii membranes by phospholipase A2 only decreased but did not abolish cholesterol uptake, an indication that glycolipids also participate in cholesterol uptake.