To investigate how intercellular coupling can be changed during Ca²⁺-overloading of ventricular muscle, we studied Ca²⁺ signals in individual cells and the histochemistry of the major gap junction channel, connexin43 (Cx43), using multicellular preparations. Papillary muscles were obtained from guinea pig ventricles and loaded with rhod-2. Sequential Ca²⁺ images of surface cells were obtained using a confocal microscope. In intact muscles, all cells showed simultaneous Ca²⁺ transients in response to field stimulation over a field of view of 0.3 x 0.3 mm². In severely Ca²⁺-overloaded muscles, obtained by high frequency stimulation in non-flowing Krebs solution, cells became less responsive to stimulation. Furthermore, non-simultaneous but serial onsets of Ca²⁺ transients were often detected suggesting a propagation delay of action potentials. The time lag of the onset between two aligned cells was sometimes as long as 100 ms. Similar lags were also observed in muscles with gap junction channels inhibited by heptanol. To investigate whether the phosphorylation state of Cx43 is affected in Ca²⁺-overloaded muscles, the distributions of phosphorylated and non-phosphorylated Cx43 were determined using specific antibodies. Most of the Cx43 was phosphorylated in the non-overloaded muscles, whereas non-phosphorylated Cx43 was significantly elevated in severely Ca²⁺-overloaded muscles. Our results suggest that the propagation delay of action potential within a small area, a few mm², can be a cause of abnormal conduction and a micro-reentry in Ca²⁺-overloaded heart. Inactivation of Na⁺ channels and inhibition of gap junctional communication may underlie the cell-to-cell propagation delay.