The purpose of the work was to develop an in vitro model for the study of lymphatic endothelium and to determine, using this model, whether or not a cytoplasmic process may be involved in transendothelial transport . Segments of canine renal hilar lymphatics were dissected clean, cannulated at both ends, and transferred to a perfusion chamber for measurement of transendothelial protein transport and for ultrastructural tracer studies . The segments were subsequently processed for light and electron microscopy. By both structural and functional criteria the lymphatics were judged to have retained their integrity. At 37°C, 36 lymphatics showed a mean rate of protein transport of 3 .51 ± 0 .45 (SEM) ug/min per cm' of lymphatic endothelium . The rate was influenced by the temperature of the system, being significantly reduced by 49% ± 4.8, 31% ± 5.3, and 29% ± 3 .9 when the temperature was lowered to 4°, 24°, and 30°C, respectively . When the temperature was raised to 40°C, the rate was significantly increased by 48% ± 12.2. The vesicular system and the intercellular regions in vessels with increased or reduced rates of transport were analyzed quantitatively to ascertain whether the rate changes could be correlated with ultrastructurally demonstrable changes in either of these postulated pathways . No significant changes in junctional or vesicular parameters were found between the control lymphatics and those perfused at 24°, 30°, and 40°C . At 4°C, the temperature at which the rate of protein transport was maximally reduced, vesicular size decreased, and the number of free cytoplasmic vesicles increased, whereas the number associated with the abluminal and luminal surfaces decreased . We concluded that isolated perfused lymphatic segments transport protein at a relatively constant rate under control conditions, and that this transendothelial transport comprises both temperaturedependent and temperature-independent mechanisms . The findings were considered in terms of the different theories of lymph formation and were interpreted as providing support for the vesicular theory .Lymph, by definition, is formed from that component of the interstitial fluid that moves across the endothelium of lymphatic vessels. However, the pathways taken and the forces that control this fluid movement are poorly understood and therefore have become the subjects of considerable controversy. According to one theory the major pathway lies between adjacent endothelial cells (11,20,21), and hydrostatic (16) or osmotic pressure (11, 12) provides the necessary force for fluid transport. Integral to this theory are the cyclic changes that occur because lymphatic vessels are alternately compressed and then relaxed within the tissues. For instance, if hydrostatic pressure is the major force, intraluminal pressure THE JOURNAL OF CELL BIOLOGY -VOLUME 98 FEBRUARY 1984 629-640 0 The Rockefeller University Press -0021-9525/84/02/0629/12 $1 .00 must be greater than atmospheric attimes, thus causing lymph to flow downstream, and less than atmo...