The Oct-4 gene encodes a transcription factor that plays an important role in maintaining the pluripotent state of embryonic stem cells and may prevent expression of genes activated during differentiation. Although its role in maintaining embryonic stem cell pluripotency is well established, there is still little known about the binding partners that regulate its function. To identify proteins that control Oct-4 function, we used affinity chromatography on immobilized Oct-4 (POU) together with MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) MS (mass spectrometry) and isolated a novel Oct-4-interacting protein, pyruvate kinase type M2 (PKM2 or M2-PK). PKM2 is an isozyme of pyruvate kinase that is specifically expressed in proliferating cells, such as embryonic stem cells, embryonic carcinoma cells, as well as cancer cells. Oct-4 and PKM2 were co-affinity precipitated from cell extracts, and glutathione S-transferase pull-down assays revealed that the POU DNA binding domain of Oct-4 was required for interaction with PKM2. In addition, the C-terminal domain of PKM2 (amino acids 307-531) was involved in binding to Oct-4. Moreover, ectopic expression of the PKM2 enhanced Oct-4-mediated transcription. These observations indicate that the transactivation potential of the Oct-4 transcription factor is positively modulated by PKM2.