The rates of protein and lipid synthesis of Mycoplasma capricolum were essentially synchronous during growth and depended on the sterol supplement in the media increasing in the order cholesterol (0.5 microgram/ml) < lanosterol (10 microgram/ml) < lanosterol (10 microgram/ml) + cholesterol (0.5 microgram/ml) < cholesterol (10 microgram/ml). The effect of lanosterol plus low cholesterol on macromolecular synthesis was synergistic. Whereas protein and lipid synthesis were brought virtually to a halt by cholesterol starvation, DNA synthesis continued for about 8 h. Increasing the palmitate and elaidate concentrations 4-fold in the lanosterol-supplemented media raised the growth rate even in the absence of the small amount of cholesterol (0.5 microgram/ml) needed otherwise for the synergistic effect on growth. Studies of the kinetics of fatty acid uptake by resting cells showed that the apparent Km (17 microM) of oleate uptake in lanosterol-grown cells was specifically lowered to 3 microM, a value equal to that seen in cholesterol-grown cells, by the inclusion of a synergistic amount of cholesterol in the growth media. By contrast, the apparent Km for palmitate uptake was the same (2 microM) for all three cell types. The results are consistent with the membrane cholesterol serving in a dual role, one as a bulk component and another more specific function involving the regulation of unsaturated fatty acid uptake and thereby phospholipid biosynthesis.