Publisher Summary Ceramide is a product of membrane sphingolipid breakdown and has been proposed as an important mediator of apoptosis. A major source of ceramide is the hydrolysis of membrane sphingomyelin (SM) to generate ceramide and phosphorylcholine, a enzymatic reaction catalyzed by sphingomyelinase (SMase). To date, five types of SMases have been described, including the lysosomal acidic SMase (A-SMase), the cytosolic ZnZ+-dependent acidic SMase, the membrane neutral magenesium-dependent SMase (N-SMase), the cytosolic magnesium-independent N-SMase, and the alkaline SMase. Activation of SMases, especially A-SMase and N-SMase, in cells in response to growth factors, cytokines, chemotherapeutic agents, irradiation, nutrient removal, and stress conditions is believed to be involved in the regulation of cell growth, differentiation, cell cycle arrest, and programmed cell death. Therefore, determination of the activity of SMases has been a powerful tool for studying the role of ceramide in the death of cells in response to various stimuli. This chapter discusses protocols for determining the activities of neutral and acidic SMases using radiolabeled substrates.