AbstractNatriuretic peptides (NPs) are important hormones that regulate multiple cellular functions including cardiovascular physiology. In the heart, two natriuretic peptide receptors NPR1 and NPR2 act as membrane guanylyl cyclases to produce 3′,5′-cyclic guanosine monophosphate (cGMP). Although both receptors protect from cardiac hypertrophy, their effects on contractility are markedly different, from little effect (NPR1) to pronounced negative inotropic and positive lusitropic responses (NPR2) with unclear underlying mechanisms. Here we use a scanning ion conductance microscopy (SICM) approach combined with Förster resonance energy transfer (FRET)-based cGMP biosensors to show that whereas NPR2 is uniformly localised on the cardiomyocyte membrane, functional NPR1 receptors are found exclusively in membrane invaginations called transverse (T)-tubules. This leads to far-reaching CNP/NPR2/cGMP signals, whereas ANP/NPR1/cGMP signals are highly confined to T-tubular microdomains by local pools of phosphodiesterase 2. This provides a previously unrecognised molecular basis for clearly distinct functional effects engaged by different cGMP producing membrane receptors.