Ca2+-stimulated adenylyl cyclases (AC) are known to play important roles in neurons but have not previously been reported in the heart. Here we present the first evidence for selective expression of Ca2+-stimulated AC in the sino-atrial node (SAN) but not in ventricular muscle of the guinea-pig heart. The AC1 isoform of Ca2+-stimulated AC was shown to be present in SAN, both as mRNA using RT-PCR and as protein using immuno-blotting with a specific antibody. Confocal immuno-fluorescence studies detected membrane localization of AC1 in SAN cells, but no AC1 in ventricular muscle. Ca2+-stimulated AC8 may also be present in SAN. The functional importance of AC activity was investigated by monitoring activation of If (gated by hyperpolarization and regulated by cAMP, which shifts activation to more depolarized voltages). Basal activity of AC in isolated SAN myocytes was demonstrated by the observations that an inhibitor of AC activity (MDL 12330A, 10 μm) shifted activation in the hyperpolarizing direction, while inhibition of phosphodiesterases (IBMX, 100 μm) shifted If activation in the depolarizing direction. Buffering cytosolic Ca2+ with the Ca2+ chelator BAPTA (by exposure to BAPTA-AM) shifted activation of If in the hyperpolarizing direction, and under these conditions the AC inhibitor MDL had little or no further effect. The actions of BAPTA were overcome by exposure to forskolin (10 μm), a direct stimulator of all AC isoforms, to restore cAMP levels. These effects are consistent with the functional importance of Ca2+-stimulated AC, which is expected to be fundamental to initiation and regulation of the heartbeat.