The kinetics of exchange of radiolabeled cholesterol and phospholipids between Mycoplasma gallisepticum cells and an excess of small unilamellar phospholipid/cholesterol vesicles were studied using cells enriched with different phospholipid classes but the same fatty acyl content. The rates of the rapidly exchanging cholesterol and phospholipid pools were markedly slower in sphingomyelin-containing cells than in phosphatidylcholine-containing cells. The decreased rates of spontaneous cholesterol and protein-mediated phospholipid exchange from the outer leaflet of the mycoplasma membrane are attributed to stronger interactions between sphingomyelin and cholesterol than between phosphatidylcholine and cholesterol. On the other hand, an increase in cholesterol exchange rate was found on incorporation of a cyclopentano-1,3-diacyl-2-phosphatidylglycerol, which interacts less with cholesterol than does the analogous glycero-1,2-diacyl-sn-3-phosphatidylglycerol.