Although studies have shown that endothelial nitric oxide synthase (eNOS) homozygous knockout mice (eNOS^−/−) develop left ventricular (LV) hypertrophy, well compensated at least to 24 wks, uncertainty still exists as to the cardiac functional and molecular mechanistic consequences of eNOS deficiency at later time-points. To bridge the gap in existent data, we examined whole hearts from eNOS^−/− and age-matched wild-type (WT) control mice ranging in age from 18 to 52 wks for macroscopic and microscopic histopathology, LV mRNA and protein expression using RNA Dot blots and Western blots, respectively, and LV function using isolated perfused work-performing heart preparations. Heart weight to body weight (HW/BW in mg/g) ratio increased significantly as eNOS^−/− mice aged (82.2%, P < 0.001). Multi-focal replacement fibrosis and myocyte degeneration/death were first apparent in eNOS^−/− mouse hearts at 40 wks. Progressive increases in LV atrial natriuretic factor (ANF) and α-skeletal actin mRNA levels both correlated significantly with increasing HW/BW ratio in aged eNOS^−/− mice (r = 0.722 and r = 0.648, respectively; P < 0.001). At 52 wks eNOS^−/− mouse hearts exhibited basal LV hypercontractility yet blunted beta adrenergic receptor (βAR) responsiveness that coincided with a significant reduction in the LV ratio of phospholamban to sarcoplasmic reticulum Ca²⁺-ATPase-2a protein levels and was preceded by a significant upregulation in LV steady-state mRNA and protein levels of the 28 kDa membrane-bound form of tumor necrosis factor-alpha. We conclude that absence of eNOS in eNOS^−/− mice results in a progressive concentric hypertrophic cardiac phenotype that is functionally compensated with decreased βAR responsiveness, and is associated with a potential cytokine-mediated alteration of calcium handling protein expression.