β₁-Adrenergic receptor activation stimulates cardiac L-type Ca²⁺ channels via adenylyl cyclases (ACs), with AC5 and AC6 being the most important cardiac isoforms. Recently, we have identified 2′(3′)-O-(N-methylanthraniloyl)-guanosine 5′-[γ-thio-]triphosphate (MANT-GTPγS) as a potent competitive AC inhibitor. Intriguingly, MANT-GTPγS inhibits AC5 and -6 more potently than other cyclases. These data prompted us to study the effects of MANT-GTPγS on L-type Ca²⁺ currents (I_Ca,L) in ventricular myocytes of wild-type (WT) and AC5-deficient (AC5^–/–) mice by whole-cell recordings. In wild-type myocytes, MANT-GTPγS attenuated I_Ca,L stimulation following isoproterenol application in a concentration-dependent manner (control, +77 ± 13%; 100 nM MANT-GTPγS, +43 ± 6%; 1 μM MANT-GTPγS, +21 ± 9%; p < 0.05). The leftward shift of current-voltage curves was abolished by 1 μM but not by 100 nM MANT-GTPγS. In myocytes from AC5^–/– mice, the residual stimulation of I_Ca,L was not further attenuated by the nucleotide, indicating AC5 to be the major AC isoform mediating acute β-adrenergic stimulation in WT mice. Interestingly, basal I_Ca,L was lowered by 1 μM but not by 100 nM MANT-GTPγS. The decrease was less pronounced in myocytes from AC5^–/– mice compared with wild types (–23 ± 1 versus –40 ± 7%), indicating basal I_Ca,L to be partly driven by AC5. Collectively, we found a concentration-dependent inhibition of I_Ca,L by MANT-GTPγS, both under basal conditions and following β-adrenergic stimulation. Comparison of data from wild-type and AC5-deficient mice indicates that AC5 plays a major role in I_Ca,L activation and that MANT-GTPγS predominantly acts via AC5 inhibition.