ABSTRACTObjective: To characterize the effect of increases of intraluminal pressure on the spontaneous diameter oscillations of isolated lymphatics and on the maximum and minimum diameter, in the presence and absence of endothelium.Methods: Afferent lymphatics were isolated from rat iliac lymph nodes. After cannulation of both ends, lymphatics were equilibrated for 60 minutes at a pressure of 6 cm H2O, and then changes in diameter oscillations to increases in perfusion pressure (from 2–10 cm H2O) were measured. The endothelium was removed by intraluminal infusion of air. Changes in diameter were expressed as a percentage of the corresponding passive diameter obtained in Ca2+‐free solution.Results: Isolated lymphatics showed phasic spontaneous constriction and dilation (∼50 to ∼150 μm). Removal of extracellular Ca2+ abolished spontaneous diameter oscillations, and the passive diameter increased from 183.0 ± 5.3 to 205.1 ± 8.8 μm as pressure increased from 2 to 10 cm H2O. In response to increases in pressure, the normalized minimum diameter (Dmin) increased (from 30.1% ± 1.1% to 54.8% ± 1.9%) while the normalized maximum diameter (Dmax) of lymphatics did not change. With increasing pressure the amplitude of vasomotion (Dmax‐Dmin) decreased, whereas the oscillation frequency increased. Removal of endothelium significantly reduced Dmax; hence, the oscillation amplitude decreased while the frequency increased. Also, endothelium removal elicited a significant change in the slope of the pressure‐Dmax curve (from 0.01 ± 0.4 to −1.43 ± 0.34).Conclusions: Increases in intraluminal pressure greatly affect the spontaneous vasomotion of lymphatics and activate the myogenic mechanism intrinsic to the smooth muscle. In addition, endothelial factors are important to maintain adequate lymphatic vasomotion. These findings suggest that intraluminal pressure and endothelial factors can be important contributors to the tone and pumping activity of lymphatics in vivo.