In this study the in vivo effects of estradiol in regulating Na+/K+-ATPase function in rat heart was studied. Adult male Wistar rats were treated with estradiol (40 µg/kg, i.p.) and after 24 h the animals were sacrificed and the heart excised. Following estradiol administration, cardiac Na+/K+-ATPase activity, expression of the α1 subunit, and phosphorylation of the α1 subunit were significantly increased. These animals also had significantly decreased levels of digoxin-like immunoreactive factor(s). Na+ levels were also significantly reduced but to a level that was still within the normal physiological range, highlighting the ability of the Na+/K+-ATPase to balance the ionic composition following treatment with estradiol. Estradiol treated rats also showed increased phosphorylation of protein kinase B (Akt), and extracellular-signal-regulated kinase 1/2 (ERK1/2). We therefore suggest a role for Akt and/or ERK1/2 in estradiol-mediated regulation of cardiac Na+/K+-ATPase expression and activity in rat heart.