Ventricular myocardium has a complex three-dimensional structure which has previously been inferred from two-dimensional images. We describe a technique for imaging the 3D organization of myocytes in conjunction with the collagen network in extended blocks of myocardium. Rat hearts were fixed with Bouin's solution and perfusion-stained with picrosirius red. Transmural blocks from the left ventricular free wall were embedded in Agar 100 resin and mounted securely in an ultramicrotome chuck. Confocal fluorescence laser scanning microscopy was used to obtain 3D images to a depth of 60 μm in a contiguous mosaic across the surface. Approximately 50 μm was then cut off the surface of the block with an ultramicrotome. This sequence was repeated 20 times. Images were assembled and registered in 3D to form an extended volume 3800 × 800 × 800 μm3 spanning the heart wall from epicardium to endocardium. Examples are given of how digital reslicing and volume rendering methods can be applied to the resulting dataset to provide quantitative structural information about the 3D organization of myocytes, extracellular collagen matrix and blood vessel network of the heart.