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The electrophysiological properties of the hearts of women and men are different. These differences are at least partly mediated by the actions of circulating estrogens and androgens on the cardiomyocytes. Experimentally, much of our understanding in this field is based on studies focusing on ventricular tissue, with considerably less known in the context of atrial electrophysiology. The aim of this investigation was to compare the electrophysiological properties of male and female atria and assess responses to acute sex steroid exposure. Age-matched adult male and female C57BL/6 mice were anesthetized (4 % isoflurane) and left atria isolated. Atria were loaded with Di-4-ANEPPS voltage sensitive dye and optical mapping performed to assess action potential duration (APD; at 10 %, 20 %, 30 %, 50 %, and 70 % repolarization) and conduction velocity in the presence of 1 nM and 100 nM 17β-estradiol or testosterone. Male and female left atria demonstrated similar baseline action potential duration and conduction velocity, with significantly greater APD70 spatial heterogeneity evident in females. 17β-estradiol prolonged action potential duration in both sexes – an effect that was augmented in females. Atrial conduction was slowed in the presence of 100 nM 17β-estradiol in both males and females. Testosterone prolonged action potential duration in males only and did not modulate conduction velocity in either sex. This study provides novel insights into male and female atrial electrophysiology and its regulation by sex steroids. As systemic sex steroid levels change and intra-cardiac estrogen synthesis capacity increases with aging, these actions may have an increasingly important role in determining atrial arrhythmia vulnerability.