Prostaglandin E₁ (PGE₁) has cardioprotective effects on the ischemic-reperfused heart. To clarify the mechanisms underlying the protective action of PGE₁ on myocardium, we examined the effect of PGE₁ on the L-type Ca²⁺ current (I_Ca) using single atrial cells from rabbits. PGE₁ did not show a significant effect on basal I_Ca but inhibited the I_Ca prestimulated by isoproterenol (Iso, 30 nM). This inhibition was concentration dependent (EC₅₀ = 0.027 µM). Both sulprostone, a specific PGE receptor subtype (EP₁ and EP₃) agonist, and 11-deoxy-PGE₁, an EP₃ agonist, inhibited the Iso-stimulated I_Ca, similar to PGE₁. Pretreatment with pertussis toxin (PTX) abolished the PGE₁ inhibition of I_Ca. Both the application of forskolin plus IBMX and intracellular dialysis with 8-bromoadenosine 3',5'-cyclic monophosphate eliminated the effect of PGE₁. PGE₁ did not show any further inhibition of I_Ca when the effect of Iso was almost fully antagonized by acetylcholine. Methylene blue (guanylate cyclase inhibitor), KT-5823 (cGMP-dependent protein kinase inhibitor), and erythro-9-(2-hydroxy-3-nonyl)adenine (type II phosphodiesterase inhibitor) did not significantly change the inhibitory effect of PGE₁. These findings suggest that 1) PGE₁ inhibits Iso-stimulated I_Ca by binding to the EP₃ receptor and 2) the PTX-sensitive and cAMP-dependent pathway is involved in the PGE₁ inhibition of I_Ca, but the nitric oxide-cGMP-dependent pathway is not. The PGE₁-induced antiadrenergic effect shown in this study may contribute to the PGE₁ protection of myocardium against ischemia.