We have investigated whether raft lipids of Madin-Darby canine kidney (MDCK) cells play any role in microvilli maintenance using a combination of atomic force microscopy (AFM) and laser scanning confocal microscopy. MDCK cells were treated to reduce the amount of sphingolipids, cholesterol, or both and subsequently imaged, in buffer solution, using AFM. It was observed that inhibition of either sphingolipid or cholesterol biosynthesis led to a reduction in the number of microvilli on the surface of MDCK cells. However, this effect was not uniform across the monolayer, with some cells resembling those in untreated controls. The subsequent extraction of cholesterol from cells grown in the presence of inhibitors led to a further reduction in microvilli on the surface of the cells and, in some cases, resulted in monolayers devoid of full length microvilli. Significantly, smaller spikes were observed on the surface of the smoother cells.