We studied the effects and mechanisms of action of NaHS, an H2S donor, on isolated phenylephrine-precontracted bovine mesenteric lymph nodes. NaHS induced concentration-dependent relaxation of lymph nodes. Removal of the endothelium reduced, but did not abolish the relaxing effect of NaHS. The relaxing effect was reduced by NO synthase inhibitor L-NAME, soluble guanylate cyclase inhibitor ODQ, ATP-sensitive K+ channel blocker glibenclamide, and a combination charybdotoxin+apamin (blockers of small- and intermediate-conductance Ca2+-activated K+ channels). Thus, the relaxing effect of H2S on lymph nodes is mediated by several parallel mechanisms. H2S induces relaxation of LN and modulates the rate of lymph transport, thereby affecting the development of immune processes in the body.