Pressure-overload hypertrophy results in downregulation of the sarcoplasmic reticulum Ca(2+)-ATPase pump encoding SERCA2 gene that regulates Ca(2+) uptake and myocardial relaxation. We previously characterized a proximal promoter region containing four Sp1 element consensus sequences (-284 to -72 base pairs (bp)) that was responsible for pressure-overload-induced transcriptional regulation. The purpose of the present study was to determine which of the Sp1 sites was responsible for the downregulation of SERCA2 gene transcription under pressure overload. Using an in vivo direct gene transfer assay, SERCA2 gene transcriptional activity was measured under pressure overload. Site-directed mutagenesis of the four Sp1 sites (I-IV) in the SERCA2 gene promoter (-284 to -72 bp) was performed. Wild-type and Sp1 mutant-luciferase reporter constructs were injected into the left-ventricular apices of pressure overload or sham-operated rats, and Sp1 mRNA and SERCA2 gene-luciferase activity was measured sequentially from 3 to 14 d after surgery. At 5 d, Sp1 mRNA in the pressure-overload rats increased to 124 +/- 7% of sham group levels, and pressure-overload-induced SERCA2 transcriptional activity was 15 +/- 4% of sham group when all four Sp1 sites remained intact. Mutation of the Sp1 mutant sites I (-196 to -191 bp) and III (-118 to -113 bp) blocked the inhibitory effect of pressure overload and resulted in SERCA2 gene transcriptional activity of 54 +/- 15% and 56 +/- 7% of sham group, respectively. We conclude that the pressure-overload-induced decrease in SERCA2 mRNA is mediated by Sp1 sites I and III.