We investigated whether in the isolated perfused rat heart acute pressure overload may affect the expression of genes involved in calcium homeostasis, namely sarcolemmal L-type Ca2+ channel, Na+/Ca2+ exchanger, sarcoplasmic reticulum Ca2+-ATPase, phospholamban, and ryanodine receptor. Hearts were subjected to 210 min of perfusion under the following conditions: (i) standard working heart perfusion with preload and afterload set at 20 and 100 cm, respectively; (ii) working heart perfusion at high afterload (180 cm); (iii) retrograde infusion of St. Thomas' Hospital cardioplegic solution. In all models gene expression was determined by RT-PCR. Significant decrease in the expression of the sarcoplasmic reticulum Ca2+-ATPase gene was observed in the high afterload group. No significant change in the expression of any other gene was observed in any group. The reported effect was not detected after 60 min of perfusion, and it was blunted in the presence of the protein kinase C inhibitor chelerythrine, while the calcineurin inhibitor cyclosporin A was ineffective. In conclusion, the sarcoplasmic reticulum Ca2+-ATPase gene is downregulated after short-term (210 min) perfusion at high afterload, possibly through a protein kinase C-dependent pathway. This mechanism might play a relevant pathophysiological role in the response to pressure overload and in the development of hypertrophy.