Via the Na/Ca and Na/H exchange, intracellular Na concentration ([Na]_i) is important in regulating cardiac Ca and contractility. Functional data suggest that [Na]_i might be heterogeneous in myocytes that are not in steady state, but little direct spatial information is available. Here we used two-photon microscopy of SBFI to spatially resolve [Na]_i in rat ventricular myocytes. In vivo calibration yielded an apparent K_d of 27±2mM Na. Similar resting [Na]_i was found using two-photon or single-photon ratiometric measurements with SBFI (10.8±0.7 vs. 11.1±0.7mM). To assess longitudinal [Na]_i gradients, Na/K pumps were blocked at one end of the myocyte (locally pipette-applied K-free extracellular solution) and active in the rest of the cell. This led to a marked increase in [Na]_i at sites downstream of the pipette (where Na enters the myocyte and Na/K pumps are blocked). [Na]_i rise was smaller at upstream sites. This resulted in sustained [Na]_i gradients (up to ∼17 mM/120μm cell length). This implies that Na diffusion in cardiac myocytes is slow with respect to trans-sarcolemmal Na transport rates, although the mechanisms responsible are unclear. A simple diffusion model indicated that such gradients require a Na diffusion coefficient of 10–12μm²/s, significantly lower than in aqueous solutions.