It was been shown, that 4-Chloro-Diazepam (4-Cl'-DZP), an inhibitor of the mitochondrial Benzodiazepine Receptors (mBzR) has cardio-protective effects against the development of arrhythmia during ischemia and reperfusion. The aim of present study was to investigate, whether inhibition of mBzR protects cardiomyocytes against the development of Ca²⁺-oscialltions and hypercontracture at the beginning of reperfusion and to analyze, whether this 4-Cl'-DZP-mediated protection is due to affecting the mitochondrial permeability transition pores (MPTP). Isolated adult rat cardiomyocytes were superfused anoxically (60 min; 100% N₂; no glucose; pH 6.4) and then reperfused with a normoxic buffer (20 min; 21% O₂; 2.5 mM glucose; pH 7.4). In addition to cell length measurement, cytosolic Ca²⁺ and MPTP-opening were measured by the fluorescence indicators Fura-2 and Calcein. The mBzR inhibitor, 4-Cl'-DZP (10 mM), the activator of the mBzR, N,N-Dihexyl-2-(4-fluorophenyl)indole-3-acetamide (FGIN; 4,6 mM) or the direct inhibitor of the MPTP, Cyclosporine A (Csp A; 500 nM) were applied during reperfusion. Application of 4-Cl'-DZP during reperfusion markedly reduced the development of cytosolic Ca²⁺-oscillations (Ca²⁺-amplitude in the 5^th min of reperfusion [a.u.]: control: 9.94±1.83; 4-Cl'-DZP: 2.26±0.68; n>=12, p<0.05 for all further measurements), (Ca²⁺-oscillations in the 5^th min of reperfusion [min^-1]: control: 55.85±10.57; 4-Cl'-DZP: 19.34± 4.50). Furthermore the extent of hypercontracture was also significantly reduced by 4-Cl'-DZP (cell length at the end of reperfusion [% of end-anoxic length: control: 51.43±1.40; 4-Cl'-DZP: 62.51±2.50). The reperfusion-induced loss of mitochondrial Calcein, which indicates the MPTP-opening, was markedly reduced either by 4-Cl'-DZP or CspA, but augmented by FGIN (Mitochondrial Calcein fluorescence [% of end-anoxic fluorescence]: control: 60.95±3.59; 4-Cl'-DZP: 74.98±2.44; Csp A: 74.64±3.79; FGIN: 48.82±2.57). The data of present study show, that inhibition of the mBzR protects cardiomyocytes against the development of Ca²⁺-oscialltions and hypercontracture at the beginning of reperfusion. This protection against reperfusion injury is mediated via inhibition of the MPTP.