An imaging system for di-4-ANEPPS (4-[-[2-(di-n-butylamino)-6-naphthylvinyl] pyridinium]) voltage-sensitive dye recordings has been adapted for recording from an in vitro mouse heart preparation, which consists of both atria in isolation. This approach has been used to study inter- and intra-atrial activation and conduction and to monitor action potential durations (APD) in the left and right atrium. The findings from this study confirm some of our previous findings in isolated mouse atrial myocytes, and demonstrate that the electrophysiological properties of mouse atria closely resemble those of larger mammals. Specifically, we made the following observations: 1) Activation in mouse atria originates in the sinoatrial node, spreads into the right atrium, and after a delay into the left atrium. 2) APD in the left atrium is shorter than in the right atrium. 3) Sites in the posterior walls have longer APDs than sites in the atrial appendages. 4) Superfusion of this preparation with 4-aminopyridine (4-AP) and tetraethylammonium (TEA) resulted in increases in APD, consistent with their inhibitory effects on the K⁺ currents known to be expressed in mouse atria. 5) The muscarinic agonist carbachol (CCh) shortened APD in all areas of the preparation, except in the left atrial appendage in which CCh had no statistically significant effect on APD. These results validate a new approach for monitoring activation, conduction and repolarization in mouse atria and demonstrate that the physiological and pharmacological properties of mouse atria are sufficiently similar to those of larger animals to warrant further studies using this preparation.