Many effects of cAMP on cardiac myocytes can only be explained in terms of microdomain signaling. Enzymes that make and break cAMP are involved in the formation of microdomains, and their role in cAMP signaling has been studied extensively. The precise geometry of microdomains depends critically on cAMP diffusion, but this is poorly understood. Earlier estimates have postulated fast cAMP diffusion, which argues against the microdomain hypothesis. Accurate determination of cAMP diffusivity requires high-resolution cAMP sensors, robust methods to evoke cAMP gradients, and appropriate protocols and analyses to dissect true diffusivity from the complex diffusion/reaction dynamics of cAMP. [cAMP] dynamics in the cytoplasm of adult rat ventricular myocytes were imaged using a fourth generation genetically-encoded FRET-based sensor. Myocytes were enzymatically isolated from adult male Sprague-Dawley rats. Cells were infected adenovirally with the construct for H187, the cAMP sensor. The [cAMP]-response to addition and removal of isoproterenol (ISO; β-adrenoceptor agonist) quantified the rates of cAMP synthesis and degradation. To obtain a read-out of DcAMP, a stable [cAMP] gradient was generated using a microfluidic device which delivered agonist to one half of the myocyte only. After accounting for phosphodiesterase activity, a [cAMP] gradient evoked by regional exposure to 1µM ISO produced an estimate of DcAMP of 35±3.4 µm2/s (n=11/4), an order of magnitude slower than cAMP diffusivity in water (444 µm2/s). Diffusivity was not dependent on the amount of cAMP generated (DcAMP measured with 10nM ISO was 32±8.7 µm2/s; n=15/4; P=0.78 vs 1µM ISO). Saturating cAMP-binding sites with the cAMP analogue 6-Bnz-cAMP (5µM, delivered as AM-ester) did not accelerate DcAMP (29±7.1 µm2/s, n=11/3; P=0.45 vs 1µM ISO) arguing against a role for buffering in restricting cAMP mobility. Molecules that are chemically-unrelated to cAMP but of comparable molecular weight (fluorescein, MagFluo4) had similar diffusivity when measured using fluorescence recovery after photobleaching (FRAP; fluorescein, 37.0±4.5 µm2/s, n=8/3; P=0.73 vs DcAMP; MagFluo4, 37.9±4.4 µm2/s n=8/3; P=0.66 vs DcAMP) suggesting that restricted mobility relates to a common physical barrier to diffusion consistent with tortuosity. Tortuosity was greater in adult myocytes compared to rat neonatal myocytes as measured by calcein diffusivity. Dcalcein (normalised to Dcalcein in pure water) was higher in neonatal myocytes (0.29±0.04, n=6/4 vs 0.13±0.01, n=16/3; P<0.05), in agreement with the 2.5-fold greater non-mitochondrial space in neonatal cytoplasm compared to adult myocytes (17±1.3, n=9/3 vs 44±1.2, n=12/3; P<0.05 determined by TMRE staining). In adult cardiac myocytes, tortuosity due to physical barriers, notably mitochondria, restricts cAMP diffusion to 32 µm2/s; a level that is more compatible with microdomain signaling.