Sphingolipid-metabolizing enzymes control the dynamic balance of the cellular levels of bioactive lipids, including the proapoptotic compound ceramide and the proliferative compound sphingosine 1-phosphate. Accumulating evidence indicates that sphingosine kinase (SK) plays a pivotal role in regulating tumor growth and that SK can act as an oncogene. Despite the importance of SK for cell proliferation, pharmacological inhibition of SK is an untested means of treating cancer because of the current lack of nonlipid inhibitors of this enzyme. To further assess the involvement of SK in human tumors, levels of RNA for SK in paired samples of cDNA prepared from tumors and normal adjacent tissue were analyzed. Expression of SK RNA was significantly elevated in a variety of solid tumors, compared with normal tissue from the same patient. To identify and evaluate inhibitors of SK, a medium throughput assay for recombinant human SK fused to glutathione S-transferase was developed, validated, and used to screen a library of synthetic compounds. A number of novel inhibitors of human SK were identified, and several representative compounds were characterized in detail. These compounds demonstrated activity at sub- to micromolar concentrations, making them more potent than any other reported SK inhibitor, and were selective toward SK compared with a panel of human lipid and protein kinases. Kinetic studies revealed that the compounds were not competitive inhibitors of the ATP-binding site of SK. The SK inhibitors were antiproliferative toward a panel of tumor cell lines, including lines with the multidrug resistance phenotype because of overexpression of either P-glycoprotein or multidrug resistance phenotype 1, and were shown to inhibit endogenous human SK activity in intact cells. Furthermore, each inhibitor induced apoptosis concomitant with tumor cell cytotoxicity. Methods for the synthesis of a series of aurone inhibitors of SK were established, and a prototypical dihydroxyaurone was found to have moderate antitumor activity in vivo in the absence of overt toxicity to the mice. These compounds are the first examples of nonlipid inhibitors of SK with in vivo antitumor activity and so provide leads for additional development of inhibitors of this important molecular target.