1. Intracellular microelectrode recordings were performed to investigate the membrane K+ conductances involved in smooth muscle hyperpolarization of lymphatic vessels in the guinea-pig mesentery. 2. Nitric oxide (NO), released either by the endothelium after acetylcholine (ACh; 10 microM) stimulation or by sodium nitroprusside (SNP; 50-100 microM), hyperpolarized lymphatic smooth muscle. These responses were inhibited with the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazole [4,3-a]quinoxalin-1-one (ODQ, 10 microM). 3. ACh and SNP-induced hyperpolarizations were inhibited (by about 90%) upon application of the ATP-sensitive K+(K(ATP)) channel blocker, glibenclamide (10 microM), or with 4-aminopyridine (2.5 mM), but were not affected by the Ca2+-activated K+ channels blocker, penitrem A (100 nM). 4. Hyperpolarization caused by the K+ channel opener, cromakalim (0.1-10 microM), isoprenaline (0.1 microM) or forskolin (0.5 microM) were all significantly blocked by glibenclamide. 5. Hyperpolarization evoked by ACh and SNP were inhibited with N-[2-(p-bromociannamylamino)-ethyl]-5-isoquinolinesulfonamide-dich loride (H89, 10 microM), suggesting the involvement of cyclic AMP dependent protein kinase (PKA). 6. These results suggest that K(ATP) channels play a central role in lymphatic smooth muscle hyperpolarization evoked by a NO-induced increase in cyclic GMP synthesis, as well as by beta-adrenoceptor-mediated production of cyclic AMP. Interestingly, both pathways lead to K(ATP) channels opening through the activation of PKA.