Abstract Background Studying cardiac electrophysiology in isolated perfused beating murine hearts is a well-established method. The ranges of normal values for left atrial (LA) action potential durations (LA-APD), activation times (LA-AT) and effective refractory periods (atrial ERP) in murine wildtype (WT) are not well known. Purpose This study aimed to establish reference values for LA-APD, LA-AT and atrial ERP and to identify the influence of genetic background, sex and age on these electrophysiological parameters in WT mice. Method We combined results from isolated beating heart Langendorff experiments carried out in WT mice between 2005 and 2019 using an octopolar catheter inserted into the right atrium and a monophasic action potential electrode recording from the LA epicardium. Electrophysiological parameters (LA-APD at 50%, 70%, 90% repolarization (APD50, APD70, APD90), LA-AT and atrial ERP) at different pacing cycle lengths (PCL) were summarized. We analysed effects of PCL, genetic background, age, gender, heart weight to body weight ratio (HW/BW), LA weight to body weight ratio (LAW/BW) as well as coronary flow and temperature as experimental conditions. Results Electrophysiological parameters from 222 isolated hearts (114 female, mean age 6.6±0.25 months, range 2.47–17.7 months) of different backgrounds (77 C57BL/6, 23 FVB/N, 33 MF1, 69 129/Sv and 20 Swiss agouti) were combined. Coronary flow rate, flow temperature and start of isolation to cannulation time were constant experimental conditions over the timespan of experiments. LA-APD was longer while LA-AT decreased with longer PCL throughout all genetic backgrounds (Figure 1A). Genetic background showed strong effects on all electrophysiological parameters. LA-APD70 and atrial ERP were significantly shorter in Swiss agouti background compared to others. LA-APD70 was also significantly prolonged in 129/Sv background compared to MF1 (Figure 1B). LA activation was delayed in 129/Sv compared to other backgrounds (Figure 1C). Atrial ERP was longer in FVB/N compared to other backgrounds. Atrial ERP was also significantly prolonged (+ 3.4 ms, + 13.5%) in female mice compared to males (Figure 1D). Age effects were compared in groups. Atrial ERP was significantly longer in mice younger than 3 months compared to older mice (Figure 1E). Conclusion This dataset summarises left atrial electrophysiological parameters in the beating mouse heart and can serve as a reference for design and interpretation of electrophysiological experiments in murine models of commonly used genetic backgrounds. We demonstrate that PCL, genetic background, age and gender affect atrial electrophysiological parameters. Awareness of these will support successful experimental design. Figure 1 Funding Acknowledgement Type of funding source: Public grant(s) – EU funding. Main funding source(s): This work was partially supported by the European Commission (grant agreements no. 633196 [CATCH ME]) to LF and PK, Deutsche Forschungsgemeinschaft DFG FA413, British Heart Foundation (FS/13/43/30324 to LF and PK; AA/18/2/34218 to LF and PK).The Institute of Cardiovascular Sciences has received the British Heart Foundation (BHF) Accelerator Award (AA/18/2/34218). JO has received financial support for abroad studies within his scholarship of the Studienstiftung des deutschen Volkes (German Academic Scholarship Foundation).