A new preparation is described for the study of the microcirculation of the rat diaphragm by in vivo microscopy. After midline laparotomy, the abdominal site of the diaphragm muscle was exposed. The rat was mechanically ventilated and placed in the Trendelenburg position, thus allowing a microscope placed on a three-dimensional articulated system to be set up perpendicular to the diaphragm. The diaphragm was then transilluminated by inserting fiberoptic microprobes into the thorax cavity by thoracotomy in the fifth intercostal space. This preparation allowed us to describe the morphological characteristics of the arteriolar network in vivo. As regards the venular network, two parts were distinguished: one part collects the blood of the external half of the diaphragm, runs roughly parallel to the arterioles, and converges toward the internal mammary and intercostal veins; the other part collects the blood of the central half of the diaphragm and converges on the central venous arcade along its central tendon. Anastomotic channels between these two parts were observed, as well as spontaneous inversion of the direction of the blood flow, indicating the presence of unsteady pressure gradients in some branches of the venular network. Capillary density was also studied by measuring intercapillary distance, whose mean value was 21.43 +/- 0.67 microns. No differences in intercapillary distance were found between the external and central parts of the diaphragm. In conclusion, we describe a preparation which allowed us to study the diaphragmatic microcirculation for at least 2 hr under good hemodynamic conditions. The study of this specific microcirculation is important because the diaphragm's metabolism and functions are specific and because it is essential to life that its perfusion should be adapted to its specific metabolic requirements.