The first enzyme of carnitine biosynthesis is the mitochondrial 6-N-trimethyllysine dioxygenase, which converts 6-N-trimethyllysine to 3-hydroxy-6-N-trimethyllysine. Using progressive membrane solubilization with digitonin and protease protection experiments, we show that this enzyme is localized in the mitochondrial matrix. Latency experiments with intact mitochondria showed that 3-hydroxy-6-N-trimethyllysine formation is limited by 6-N-trimethyllysine transport across the mitochondrial inner membrane. Because the subsequent carnitine biosynthesis enzymes are cytosolic, after production, 3-hydroxy-6-N-trimethyllysine must be transported out of the mitochondria by a putative mitochondrial 6-N-trimethyllysine/3-hydroxy-6-N-trimethyllysine transporter system. This transport system represents an additional step in carnitine biosynthesis that could have considerable implications for the regulation of carnitine biosynthesis.