Severe ketoacidosis induces heart failure and cardiac arrest, but its mechanism is unknown. Recently, hydroxy-carboxylic acid receptor 2 (HCA(2)) was found to be a receptor for a ketone body, β-hydroxybutyric acid (BHB), and is coupled with Gi-GTP binding protein. HCA(2) expression was reported in the guinea pig heart. Therefore, using guinea pig cardiac myocytes, we investigated effects of BHB on L-type Ca(2+) current pre-augmented with β-adrenoceptor agonist, isoproterenol under the whole-cell voltage clamp. BHB significantly reduced the Ca(2+) current pre-augmented with isoproterenol. The effect of BHB was concentration dependent with IC(50) of 1.1 mM. Nicotinic acid (NA), another ligand for HCA(2), also exerted an effect on the Ca(2+) current similar to that of BHB. The effects of BHB and NA were reduced by a specific Gi inhibitor, pertussis toxin in the pipette solution. Our results suggest that BHB activates Gi-coupled signal transduction pathway via HCA(2) in guinea pig cardiac myocytes. The HCA(2)-mediated signal transduction may be associated with ketoacidosis-induced cardiac suppression.