TRPM3, a member of the melastatin-like transient receptor potential channel subfamily (TRPM), is predominantly expressed in human kidney and brain. TRPM3 mediates spontaneous Ca2+ entry and nonselective cation currents in transiently transfected human embryonic kidney 293 cells. Using measurements with the Ca2+-sensitive fluorescent dye fura-2 and the whole-cell patch-clamp technique, we found that D-erythro-sphingosine, a metabolite arising during the de novo synthesis of cellular sphingolipids, activated TRPM3. Other transient receptor potential (TRP) channels tested [classic or canonical TRP (TRPC3, TRPC4, TRPC5), vanilloid-like TRP (TRPV4, TRPV5, TRPV6), and melastatin-like TRP (TRPM2)] did not significantly respond to application of sphingosine. Sphingosine-induced TRPM3 activation was not mediated by inhibition of protein kinase C, depletion of intracellular Ca2+ stores, and intracellular conversion of sphingosine to sphingosine-1-phosphate. Although sphingosine-1-phosphate and ceramides had no effect, two structural analogs of sphingosine, dihydro-D-erythro-sphingosine and N,N-dimethyl-D-erythro-sphingosine, also activated TRPM3. Sphingolipids, including sphingosine, are known to have inhibitory effects on a variety of ion channels. Thus, TRPM3 is the first ion channel activated by sphingolipids.