Substance P (SP) is a neuropeptide associated with sensory innervation of lymphoid tissue and a suspected modulator of lymphatic function in inflammation. Only a few studies have examined the effects of SP on lymphatic contraction and it is not clear to what extent SP acts directly on lymphatic muscle and/or endothelium or indirectly through changes in intraluminal filling pressure secondary to increases in capillary permeability / filtration. We tested the effects of SP on the spontaneous contractions of rat isolated mesenteric lymphatic vessels under isometric and isobaric conditions, hypothesizing that low concentrations would stimulate lymphatic pumping by enhancing lymphatic muscle contraction in a manner complementary to the effect of increased preload. Under isometric conditions, SP (10 nM) dramatically enhanced lymphatic chronotropy and inotropy. Unlike guinea pig lymphatics, SP actions were not blocked by COX or PLA₂ inhibition. In the absence of SP, ramp increases in isometric preload resulted in ~1.6x increases in contraction amplitude (AMP) and ~1.7x increases in frequency (FREQ). SP increased FREQ by ~2.4x, AMP by ~1.9x, and the AMP-FREQ product (AFP) by ~3.5x. Under isobaric conditions, pressure elevation from 0.5 to 10 cmH₂O in the absence of SP decreased AMP by ~0.6x and increased FREQ by ~1.8x. SP caused a modest increase in AMP, a robust increase in FREQ at all pressures, and shifted the AFP-pressure relationship upward and leftward. Therefore, SP has substantial positive inotropic and chronotropic effects on rat lymphatic muscle, improving pump efficiency independent of the effects of preload, and broadening of the working range of the lymphatic pump.