Sinoatrial nodal cells (SANC) express Ca2+-activated adenylate cyclase (AC) isoforms that generate a high basal level of cAMP-mediated, protein kinase A (PKA)-dependent Ca2+ cycling protein phosphorylation, resulting in spontaneous rhythmic Ca2+ oscillations that ignite the surface membrane to generate rhythmic APs, i.e. pacemaker automaticity. Differences in basal phosphodiesterase (PDE) and AC activities and in PDE:AC activity within protein microenvironments in SANC are a potential mechanism for compartmental regulation of cAMP levels, leading to local differences in the effectiveness of cAMP signaling within these microdomains. The present study measured the AC activity and PDE activity in SANC in detergent-resistant microdomains (DRM) of SANC lysates to determine the PDE and AC activities in lipid raft gradients indexed by GM-1 and caveolin-3 immunolabeling. The microdomain Ca2+ dependence of AC activity, and the relative abundance of microdomain PDE types, based upon the effects of specific PDE inhibitors, were also determined. Under the conditions of our assay, PDE and AC activities are nearly identically matched in the fractions that contain higher densities of lipid raft markers. As the lipid raft density decreases below a threshold the PDE:AC activity ratio becomes increased: from 10 to more then 200 fold , depending upon the Ca2+ milieu, while neither the relative extent of AC activation by different Ca2+ significantly varied among the different microenvironments, the Ca2+ milieu in lipid raft-rich fractions affects the matching of PDE to AC activities. The most optimal milieu for cAMP production and survival is when the Ca2+ is 1 M, and the lipid raft marker density is high;when Ca2+ is reduced to 200 nM or heavily buffered by BAPTA, however, PDE activity exceeds that of AC, even in lipid raft-rich fractions. The very high PDE activity and lower AC activity in non-lipid raft domains favors degradation of cAMP, not only maintaining a reduced local level of cAMP production, but also creating a functional barrier for diffusion of cAMP from lipid rafts into non-lipid raft domains or into the cytosol.