Fluid dynamic forces have substantial effects on the movement of lymph and activity of lymph vessels. The effect of increases in intraluminal flow on spontaneous pumping activity of isolated collecting lymphatics has not yet been characterized in a condition in which the intraluminal pressure is constant. Thus, in afferent lymph microvessels isolated from rat iliac lymph nodes, changes in maximum (D_max) and minimum (D_min) diameter to increases in perfusate flow were investigated in the presence of a constant perfusion pressure of 6 cmH₂O. Intraluminal flow was elicited by increases in the difference between outflow and inflow pressures (P_diff, from 0 to 6 cmH₂O). Diameters were measured by videomicroscopy. In response to increases in perfusate flow, D_max and D_min of lymphatics decreased from 157.5 ± 6.1 to 90.9 ± 5.6 µm and from 91.9 ± 5.3 to 66.3 ± 3.6 µm, respectively, whereas vasomotion frequency increased from 18.0 ± 0.7 min¹ to 23.4 ± 1.1 min¹ (at P_diff of 4 cmH₂O). Removal of extracellular Ca²⁺ abolished spontaneous diameter oscillations; under these conditions the passive diameter of lymphatics was 216.0 ± 7.1 µm and did not change in response to increases in perfusion. In the absence of endothelium, flow-induced changes in D_max, D_min, and oscillation frequency were eliminated. N-nitro-L-arginine methyl ester, an inhibitor of nitric oxide synthase, did not affect flow-induced changes in diameter of lymphatics. In contrast, indomethacin, an inhibitor of prostaglandin synthesis, or SQ-29,548, a PGH₂/thromboxane A₂ (PGH₂/TxA₂) receptor blocker, inhibited the perfusion-induced reduction of D_max and D_min of lymphatics and also the increase in the frequency of vasomotion. These findings suggest that the sensitivity of lymphatic endothelium to increases in intraluminal flow could provide an important local intrinsic mechanism for the control of lymphatic resistance by release of constrictor prostanoids PGH₂/TxA₂.