The muscular lymphatics use both phasic and tonic contractions to transport lymph for conducting its vital functions. The molecular mechanisms regulating lymphatic muscle contractions are not well understood. Based on the well-established finding that the phosphorylation of myosin light chain 20 (MLC₂₀) plays an essential role in blood vessel smooth muscle contraction, we investigated if MLC₂₀ phosphorylation (pMLC₂₀) would modulate the tonic and/or phasic contractions of lymphatic muscle. The effects of ML-7, a myosin light chain kinase (MLCK) inhibitor (1-10 µM), were tested on the contractile parameters of isolated and cannulated rat mesenteric lymphatics during their responses to the known modulators, pressure (1-5 cm H₂O) and substance P (SP, 10-7 M). Immunohistochemical and Western blot analyses of phospho-MLC₂₀ were also performed on isolated lymphatics. The results showed that: 1. Increasing pressure decreased both the lymphatic tonic contraction strength and the pMLC₂₀/MLC₂₀ ratio. 2. SP increased both the tonic contraction strength and the phosphorylation of the MLC₂₀. 3. ML-7 decreased both the lymphatic tonic contraction strength and the pMLC₂₀/MLC₂₀ ratio. 4. The increase in lymphatic phasic contraction frequency in response to increasing pressure is diminished by ML-7; however the phasic contraction amplitude was not significantly altered by ML-7 either in the absence or presence of SP. These data provide the first evidence that tonic contraction strength and phasic contraction amplitude of the lymphatics can be differentially regulated, whereby the increase in MLC₂₀ phosphorylation produces an activation in the tonic contraction without significant changes in phasic contraction amplitude. Thus, tonic contraction of rat mesenteric lymphatics appears to be MLCK-dependent.