Estrogen receptor alpha (ERalpha) is present in the heart consistent with estrogen-induced modulation of cardiac function by genomic and non-genomic mechanisms, and with estrogen-mediated cardioprotective effects. We show that, in heart from adult male rats, ERalpha is detected mainly as two distinct isoforms: (i) a approximately 66 kDa isoform with the expected mass of the classical full-length ERalpha and (ii) an additional isoform of approximately 45 kDa. Differential centrifugation separated the 66 kDa isoform into the cytosolic fraction; while the 45 kDa isoform was enriched in the membrane fraction. High-resolution confocal studies show that ERalpha is distributed in the nucleus, cytosol, and various membranes including the plasmalemma. Notoriously, ERalpha labeling was very prominent in T-tubular membranes defined by alpha-actinin staining and the intercalated disks. In the T-tubules, ERalpha degree of association to alpha-actinin depends on the distribution pattern of the receptor along the T-tubules; association is high when ERalpha pattern is "continuous," while it is low when the receptor has a discontinuous "granular" distribution. Nuclear ERalpha has a distinct trabecular distribution and it is excluded from the heterochromatin, consistent with an active transcription factor. Treatment with estrogen ( approximately 4 h) produced an overall decrease in both nuclear and non-nuclear ERalpha levels and made more evident discrete ERalpha nuclear puncta uncovering cellular mechanism(s) of short term action of estrogen in the heart. The results indicate that the levels of the cardiac ERalpha isoforms are downregulated by estrogen and are differentially distributed: the full-length ERalpha is mainly compartmentalized in the cytosol and nucleus, while the 45 kDa isoform is mainly present in membrane structures. The membrane localization of ERalpha may support the rapid effects of estrogens on heart function.