Differences in lipid composition and the effects of monolayer and suspension culture were studied in Madin-Darby canine kidney cells and their plasma membranes isolated by the Warren fluorescein-mercuric acetate technique. Unesterified cholesterol was the major neutral lipid extracted from the whole cells and plasma membranes of cultures except for suspension-grown whole cells which had triglyceride as their major neutral lipid. Triglyceride increased about 7- to 20-fold and glycerol ethers increased about 3- to 20-fold in suspension grown whole cells and their plasma membranes compared to those from monolayer cultures. Total quantities of phospholipids are similar in whole cells of both cultures. Plasma membranes from monolayer cultures have about 1.4-fold more total phospholipids than those from suspension cultures. The distribution of fatty acids in the plasma membranes and whole cells generally follow the composition of calf serum used in the culture medium. However, the phospholipids of plasma membranes from monolayer cultures had substantially higher values for C18 unsaturated fatty acids and somewhat lower values for palmitoleic acid than the values for the fatty acids in calf serum. Also, the phospholipids of suspension-grown whole cells had substantially higher values for lauric acid and lower values for C18 unsaturated fatty acids when compared to calf serum.