Background and Purpose: The aim of this study was to investigate the mechanism of a possible differential effect of Neocuproine (NC), selective Cu(1) chelator, in rat and mouse bladder tissues. Experimental Approach: Bladder function was evaluated by 1. in vitro isolated bladder strips, 2. in vitro preparations of whole bladders and 3. İn vivo cystometrogram (CMG) in rat and mouse. Selective Cu(I) and Cu(II) chelators and non-selective purinergic antagonists were examined on EFS-induced bladder contractions in isolated bladder strips. The spontaneous contraction activities of whole bladders were recorded to evaluate the amplitudes and frequencies. In CMGs experiments, the values of maximum bladder pressure during micturition and intercontraction intervals (ICI) were evaluated. Key Results: Whereas Neocuproine (NC) enhances the opposite effect on isolated bladder strips and whole bladder experiments, this opposite effect was not observed in in vivo CMG experiments in rat and mouse. NC caused a significant suppression on spontaneous contractions and baseline tonus in isolated mouse bladder tissues, whereas it caused facilitating effects in the rat. However, NC significantly decreased the ICI in both rat and mouse in CMGs. Conclusion and Implications: The effect of Neocuproine on rat and mouse bladder activity is likely to be the role of myogenic mechanisms. It is possible to trigger mechanisms associated with intracellular calcium (Ca2+) reduction and purinergic pathway, P2X purinoceptors besides P2Y purinoceptors, may have an important role in the inhibitory activity of NC on mouse bladder.