A series of experimental data point to the existence of profound diffusion restrictions of ADP/ATP in rat cardiomyocytes. This assumption is required to explain the measurements of kinetics of respiration, sarcoplasmatic reticulum loading with calcium, and kinetics of potassium ATP-sensitive channels. To be able to analyze and estimate the role of intracellular diffusion restrictions on bioenergetics, the intracellular diffusion coefficients of metabolites have to be determined. The aim of this work was to develop a practical method for determining of diffusion coefficients in anisotropic medium and to estimate the overall diffusion coefficients of fluorescently labeled ATP in rat cardiomyocytes. For that, we have extended raster image correlation spectroscopy protocols to be able to discriminate the anisotropy in diffusion coefficient tensor. Using this extended protocol, we estimated diffusion coefficients of ATP labeled with the fluorescent conjugate Alexa Fluor 647 (Alexa-ATP). In the analysis, we assumed that the diffusion tensor can be described by the two values: diffusion coefficient along the myofibril and across it. The average diffusion coefficients found for Alexa-ATP were as follows: 83±14µm²/s in longitudinal and 52±16µm²/s in transversal directions (n=8, mean±SD). Those values are 2 (longitudinal) and 3.5 (transversal) times smaller than the diffusion coefficient value estimated for surrounding solution. Such uneven reduction of average diffusion coefficient leads to anisotropic diffusion in the rat cardiomyocytes. While the source for such anisotropy is uncertain, we speculate that it may be induced by ordered pattern of intracellular structures in rat cardiomyocytes.