The alpha-ketoglutarate dehydrogenase complex (KGDHC), a control point of the tricarboxylic acid cycle, is partially inactivated in brain in many neurodegenerative diseases. Potent and specific KGDHC inhibitors are needed to probe how the reduced KGDHC activity alters brain function. Previous studies showed that succinyl phosphonate (SP) effectively inhibits muscle and Escherichia coli KGDHC [Biryukov, A. I., Bunik, V. I., Zhukov, Yu. N., Khurs, E. N., and Khomutov, R. M. (1996) FEBS Lett. 382, 167-170]. To identify the phosphonates with the highest affinity toward brain KGDHC and with the greatest effect in living cells, we investigated the ability of SP and several of its ethyl esters to inhibit brain KGDHC, other alpha-keto acid-dependent enzymes, and KGDHC in intact cells. At a concentration of 0.01 mM, SP and its phosphonoethyl (PESP) and carboxyethyl (CESP) esters completely inhibited isolated brain KGDHC even in the presence of a 200-fold higher concentration of its substrate [alpha-ketoglutarate (KG)], while the diethyl (DESP) and triethyl (TESP) esters were ineffective. In cultured human fibroblasts, 0.01 mM SP, PESP, or CESP produced 70% inhibition of KGDHC. DESP and TESP were also inhibitory in the cell system, but only after preincubation, suggesting the release of their charged groups by cellular esterases. Thus, SP and its monoethyl esters target cellular KGDHC directly, while the di- and triethyl esters are activated in intact cells. When tested on other enzymes that bind KG or related alpha-keto acids, SP had minimal effects and its two esters (CESP and TESP) were ineffective even at a concentration (0.1 mM) 1 order of magnitude higher than that which inhibited cellular KGDHC activity. The high specificity in targeting KGDHC, penetration into cells, and minimal transformation by cellular enzymes indicate that SP and its esters should be useful in studying the effects of reduced KGDHC activity on neuronal and brain function.