The three-dimensional architecture of the rat pulmonary veins was studied by light microscopy (LM) and scanning electron microscopy (SEM). For LM, the left lungs were fixed with formalin, sectioned and immunostained with an anti-alpha-smooth muscle actin (alpha-SMA) antibody in addition to conventional staining. For SEM, the specimens were fixed with glutaraldehyde and immersed in 30% KOH solution for 8 min followed by treatment of collagenase solution for more than 5 h. By LM, the smooth muscle cells stained with anti-alpha-SMA showed discontinuous, periodical thickenings of circular bundles in the wall of the venules, but they became thin and continuous in the larger vessels (or veins) that had a cardiac muscle layer on the outside. Under SEM, the smooth muscle cells formed circular-oriented bundles at constant intervals along the venules less than 100 microm in diameter. These bundles had circumferential constrictions in the lumen. The cardiac muscle cells, which appeared in large pulmonary veins of more than 100 microm, ran in a circular or oblique direction and completely surrounded the vessel wall outside of the thin continuous layer of smooth muscle cells. The muscle arrangements were considered to play a significant role in the return blood flow in rat pulmonary veins.