We endeavored to use a basic and well controlled experimental system to characterize the extent and time sequence of sarcoplasmic reticulum ATPase (SERCA) involvement in the development of cardiac hypertrophy, including transcription, protein expression, Ca²⁺ transport and cytoplasmic Ca²⁺ signaling. To this aim, hypertrophy of neonatal rat cardiac myocytes in culture was obtained following adrenergic activation with phenylephrine (PE). Micrographic assessment of myocyte size, rise of (14C)-phenylalanine incorporation and total protein expression, and increased transcription of Atrial Natriuretic Factor (ANF), demonstrated unambiguously the occurrence of hypertrophy. An early and prominent feature of hypertrophy was a reduction of the SERCA2 transcript, as determined by RT-PCR with reference to a stable marker such as Glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Reduction of Ca²⁺ ATPase protein levels and Ca²⁺ transport activity to approximately 50% of control values followed with some delay, evidently as a consequence of a primary effect on transcription. Cytosolic Ca²⁺ signaling kinetics, measured with a Ca²⁺ sensitive dye following electrical stimuli, were significantly altered in hypertrophic myocytes. However, the effect of PE hypertrophy on cytosolic Ca²⁺ signaling kinetics was less prominent than observed in myocytes subjected to drastic SERCA2 downregulation with siRNA or inhibition with thapsigargin (10 nM). We conclude that SERCA2 undergoes significant downregulation following hypertrophic stimuli, possibly due to lack of the SERCA gene involvement by the hypertrophy transcriptional program. The consequence of SERCA2 downregulation on Ca²⁺ signaling is partially compensated by alternate Ca²⁺ transport mechanisms. These alterations may contribute to a gradual onset of functional failure in long term hypertrophy.