Instrumental role of Na⁺ and Ca²⁺ influx via Na⁺/K⁺ adenosine triphosphatase (Na⁺/K⁺-ATPase) and Na⁺/Ca²⁺ exchanger 1 (NCX1) is examined in the N-Methyl-D-aspartate (NMDA) receptor-mediated pathogenesis of penumbra after focal cerebral ischemia. An experimental model of 3, 6, and 24 h focal cerebral ischemia by permanent occlusion of middle cerebral artery was developed in rats. The changes in protein expression of Na⁺/K⁺-ATPase and NCX1 as well as functional subunits of NMDA receptor 2A and 2B (NR2A and NR2B) in the penumbra were assessed using by quantitative immunoblottings. The most prominent changes of Na⁺/K⁺-ATPase (78±6%, n=4, ^* P <0.05) and NCX1 (144±2%, n=4, ^* P <0.05) in the penumbra were developed 24 h after focal cerebral ischemia. The expression of NR2A in the penumbra was significantly increased (153±9%, n=4, ^* P <0.05) whereas the expression of NR2B was significantly decreased (37±2%, n=4, ^* P <0.05) as compared with sham-operated controls 3 h after focal cerebral ischemia. However, the expression of NR2A and NR2B in the penumbra was reversed 24 h after focal cerebral ischemia (NR2A: 40±7%; NR2B: 120±16%, n=4, ^* P <0.05). Moreover, the decreased expression of neuronal nuclei (NeuN) in the penumbra was most prominent than that of glial fibrillary acidic protein (GFAP) 24 h after focal cerebral ischemia. These findings imply that intracellular Na⁺ accumulation via decreased Na⁺/K⁺-ATPase exacerbate the Ca²⁺ overload cooperated by the increased NCX1 and NR2B-containing NMDA receptor which may play an important role in the pathogenesis of the penumbra.