Increasing evidence shows a marked beneficial effect with β-blockers in heart dysfunction via scavenging reactive oxygen species. Previously we showed that chronic treatment with either timolol or propranolol possessed similar beneficial effects for heart function in male rats as age increased, whereas only timolol exerted similar benefits in female rats. Therefore, in this study, we aimed first to examine the cellular bases for age-related alterations in excitation-contraction coupling in ventricular myocytes from female rats and, second, to investigate the hypothesis that age-related changes in [Ca2+]i homeostasis and receptor-mediated system can be prevented with chronic timolol treatment. Chronic timolol treatment of 3-month-old female rats abolished age-related decrease in left ventricular developed pressure and the attenuated responses to β-adrenoreceptor stimulation. It also normalized the altered parameters of [Ca2+]i transients, decreased Ca2+ loading of sarcoplasmic reticulum and increased basal [Ca2+]i, and decreased L-type Ca2+ currents in 12-month-old female rats compared with the 3-month-old group. Adenylyl cyclase activity, b-adrenoreceptor affinity to its agonist, and b-adrenoreceptor density of the 12-month-old group are normalized to those of the 3-month-old group. Moreover, timolol treatment prevented dysfunction of the antioxidant system, including increased lipid peroxidation, decreased ratio of reduced glutathione to oxidized glutathione, and decreased activities of thioredoxin reductase and glucose-6-phosphate dehydrogenase, in the left ventricle of hearts from the 12-month-old group. Our data confirmed that aging-related early myocardial impairment is primarily related to a dysfunctional antioxidant system and impairment of Ca2+ homeostasis, which can be prevented with chronic timolol treatment.