We have used quantitative epifluorescence microscopy of fluorescent ATP to measure single-nucleotide turnover in skinned skeletal muscle fibers from mouse models of female aging and hormone treatment. Aging causes declines in muscle strength, often leading to frailty, disability, and loss of independence for the elderly. Female muscle is additionally affected by age due to reduction of ovarian hormone production with menopause. Estradiol (E-2) is the key hormonal signal to skeletal muscle in females, and strength loss is attenuated by E-2 treatment. To investigate Ey mechanisms on skeletal muscle, single fibers were isolated from sham-operated or ovariectomized (OVX) mice, with or without Ey treatment, and were incubated with 2'-(or-3')-O-(N-methylanthraniloyl) adenosine 5'-triphosphate (mantATP). We measured decay of mantATP fluorescence in an ATP-chase experiment, as pioneered by Cooke and coworkers, who unveiled a novel regulated state of muscle myosin characterized by slow nucleotide turnover on the order of minutes, termed the super-relaxed state (SRX). We detected a slow phase of nucleotide turnover in a portion of the myosin heads from sham fibers, consistent with SRX. Turnover was substantially faster in OVX fibers, with a turnover time constant for the slow phase of 65 +/- 8 s as compared to 102 +/- 7 s for sham fibers. 60-days Ey treatment in OVX mice substantially reversed this effect on SRX, while acute exposure of isolated muscles from OVX mice to E-2 had no effect. We conclude that E-2-mediated signaling reversibly regulates slow ATP turnover by myosin. Age- and hormone-related muscle functional losses may be targetable at the level of myosin structure/function for strategies to offset weakness and metabolic changes that occur with age. (C) 2014 Elsevier Inc. All rights reserved.