Tyrphostin 23 (T23) is a well-known inhibitor of protein tyrosine kinases and has been considered as potential anti-cancer drug. T23 was recently reported to acutely stimulate the glycolytic flux in primary cultured astrocytes. To investigate whether T23 also affects the tricarboxylic acid (TCA) cycle, we incubated primary rat astrocyte cultures with [U-(13)C]glucose in the absence or the presence of 100 μM T23 for 2 h and analyzed the (13)C metabolite pattern. These incubation conditions did not compromise cell viability and confirmed that the presence of T23 doubled glycolytic lactate production. In addition, T23-treatment strongly increased the molecular carbon labeling of the TCA cycle intermediates citrate, succinate, fumarate and malate, and significantly increased the incorporation of (13)C-labelling into the amino acids glutamate, glutamine and aspartate. These results clearly demonstrate that, in addition to glycolysis, also the mitochondrial TCA cycle is strongly accelerated after exposure of astrocytes to T23, suggesting that a protein tyrosine kinase may be involved in the regulation of the TCA cycle in astrocytes.