There is strong support for the view that ventricular myocardium has a laminar organization in which myocytes are grouped into branching layers separated by cleavage planes. However, understanding of the extent and functional implications of this architecture has been limited by the lack of a systematic three-dimensional (3-D) description of the organization of myocytes and associated perimysial collagen. We have imaged myocytes and collagen across the left-ventricular (LV) wall at high resolution in seven normal rat hearts using extended-volume confocal microscopy. We have developed novel reconstruction and segmentation techniques necessary for quantitative analysis of 3-D myocyte and perimysial collagen organization. The results confirm that perimysial collagen has an ordered arrangement and that it defines a laminar organization. Perimysial collagen is comprised of three distinct forms: extensive meshwork on laminar surfaces, convoluted fibers connecting adjacent layers, and longitudinal cords. While myolaminae are the principal form of structural organization throughout most of the wall, they are not seen in the subepicardium where perimysial collagen is present only as longitudinal cords.