Repeated Ca2+ depletion and repletion of short duration, termed Ca2+ preconditioning (CPC), is hypothesized to protect the heart from lethal injury after exposing it to the Ca2+ paradox (Ca2+ PD). Hearts were preconditioned with five cycles of Ca2+ depletion (1 minute) and Ca2+ repletion (5 minutes). These hearts were then subjected to Ca2+ PD, ie, one cycle of Ca2+ depletion (10 minutes) and Ca2+ repletion (10 minutes). Hearts subject to the Ca2+ PD underwent rapid necrosis, and myocytes were severely injured. CPC hearts showed a remarkable preservation of cell structure; ie, 65% of the cells were normal in CPC hearts compared with 0% in the Ca2+ PD hearts. LDH release was significantly reduced in CPC hearts compared with Ca2+ PD hearts (2.45 ±0.18 and 8.02±0.7 U · min · g−, respectively). ATP contents of CPC hearts were less depleted compared with the Ca2+ PD hearts (5.9±0.8 and 3.0±0.16 μmol/g dry weight, respectively). Addition of the adenosine A1 receptor agonist R-phenylisopropyl adenosine before and during Ca2+ PD provided protection similar to that in CPC hearts, whereas the nonselective adenosine A1 receptor antagonist, 8-(p-sulfophenyl)-theophylline, blocked the beneficial effects of CPC. CPC-mediated protection was aborted when hearts subjected to CPC were treated with pertussis toxin (the guanine nucleotide or G-protein inhibitor). The present study suggests that Ca2+ preconditioning confers significant protection against the lethal injury of Ca2+ PD in rat hearts. Cardioprotection appears to result from adenosine release during preconditioning and by Gi-protein-modulated mechanisms.