Mapping the myocardial fiber organization is important for assessing the electrical and mechanical properties of normal and diseased hearts. Current methods to determine the fiber organization have several limitations: histological sectioning mechanically distorts the tissue and is labor-intensive, while diffusion tensor imaging has low spatial resolution and requires expensive MRI scanners. Here, we utilized optical clearing, a fluorescent dye, and confocal microscopy to create three-dimensional reconstructions of the myocardial fiber organization of guinea pig and mouse hearts. We have optimized the staining and clearing procedure to allow for the nondestructive imaging of whole hearts with a thickness up to 3.5 mm. Myocardial fibers could clearly be identified at all depths in all preparations. We determined the change of fiber orientation across strips of guinea pig left ventricular wall. Our study confirms the qualitative result that there is a steady counterclockwise fiber rotation across the ventricular wall. Quantitatively, we found a total fiber rotation of 105.7 ± 14.9° (mean ± standard error of the mean); this value lies within the range reported by previous studies. These results show that optical clearing, in combination with a fluorescent dye and confocal microscopy, is a practical and accurate method for determining myocardial fiber organization. Microsc. Res. Tech., 2008. © 2008 Wiley-Liss, Inc.