1 The present study has investigated mechanisms underlying ATP-induced endothelium-independent enhancement of vasomotion in guinea-pig mesenteric lymphatic vessels. 2 Lymphatic vasomotion, vessel tone and smooth muscle [Ca 2+] i showed similar ATP concentration-response curves. 3 ATP, at 0.1 mM, caused a biphasic increase in tonic [Ca 2+] i and superimposed vasomotion-associated Ca 2+ transients. All ATP-induced [Ca 2+] i changes were abolished by incubating the smooth muscle with suramin (0.1 mM). 4 α,β-MeATP (0.1 mM) and UTP (0.1 mM) caused similar changes in [Ca 2+] i but the responses to these agonists were smaller than to ATP. 5 The actions of α,β-MeATP (0.1 mM) were inhibited by suramin (0.1 mM) and PPADS (30 μM) but not by reactive blue 2 (30 μM). 6 In the presence of α,β-MeATP (0.1 mM), the increases in tonic [Ca 2+] i and vasomotion-associated Ca 2+ transients induced by ATP (0.1 mM) were inhibited by U73122 (5 μM), CPA (20 μM) and heparin, whereas U73343 (5 μM) and pre-treatment with PTx (100 ng ml -1) had no significant effects. 7 Depletion of the intracellular stores with CPA (20 μM) caused an increase in [Ca 2+] i, which was not blocked by desensitization of P 2X receptors with α,β-MeATP. 8 The data indicate that ATP, at relatively high concentrations increases lymphatic smooth muscle [Ca 2+] i and vasomotion through activation of P 2X1 and P 2Y2 purinoceptors present on lymphatic smooth muscle. The increase in [Ca 2+] i is likely to result from Ca 2+ release from inositol-1,4,5-trisphosphate-sensitive stores as well as Ca 2+ influx through store-operated channels and P 2X-gated channels.