Histone deacetylase 6 (HDAC6) controls acetylation of a number of cytosolic proteins, most prominently tubulin. Tubacin is a small molecule inhibitor of HDAC6 selected for its selective inhibition of HDAC6 relative to other histone deacetylases. For this reason it has become a useful pharmacological tool to discern the biological functions of HDAC6 in numerous cellular processes. The interest of this laboratory is in the function and regulation of sphingolipids, a family of lipids based on the sphingosine backbone. Sphingolipid biosynthesis is initiated by the rate limiting enzyme serine palmitoyltransferase (SPT). Sphingolipids have critical and diverse functions in cell survival, apoptosis, intra- and intercellular signaling, and in membrane structure. In the course of examining the role of HDAC6 in the regulation of sphingolipid biosynthesis we observed that tubacin strongly inhibited de novo synthesis whereas HDAC6 knockdown very moderately stimulated synthesis. We resolved these seemingly contradictory results by demonstrating that, surprisingly, tubacin is a direct inhibitor of SPT activity in permeabilized cells. Furthermore tubacin inhibits de novo sphingolipid synthesis in intact cells at doses commonly used to test HDAC6 function and does so in an HDAC6-independent manner. Niltubacin is a chemical analog of tubacin which lacks tubacin's HDAC6 activity, and so is often used as a control for off-target effects of tubacin. We find that niltubacin is inactive in the inhibition of sphingolipid biosynthesis, and so does not serve to distinguish the inhibitory effects of tubacin on HDAC6 from those on sphingolipid biosynthesis. These results indicate that caution should be used in the use of tubacin to study the role of HDAC6. (C) 2014 Elsevier Inc. All rights reserved.