Although cholesterol accumulation in tumors was first reported in the early20th century, the mechanistic implications of this observation are stillobscure. Here we report that caveolin-negative human prostate cancer (LNCaP) cells contain cholesterol-rich lipid rafts that mediate epidermal growth factor (EGF)-induced and constitutive signaling through the Akt1 serine-threonine kinase. EGF receptor and Akt1 phosphorylation were inhibited and autonomous cell survival was reduced when the rafts were disrupted. Reconstitution of the rafts with cholesterol restored EGF receptor→Akt1 axis signaling and cytoprotection from a phosphoinositide 3-kinase-dependent apoptotic signal. These results suggest that cholesterol present in membrane microdomains is a prominent mediator of survival in prostate cancer cells.