The mode of action of atrial natriuretic peptide (ANP) on bovine mesenteric lymphatics was investigated by recording isometric tensions in isolated cylindrical segments. ANP in concentrations from 5 to 30 ng/ml caused dose-related decreases in the rhythm and amplitude of spontaneous contractions. No tachyphylaxis was observed in the ANP-induced responses in lymph vessels. Addition of ANP in a low concentration ranging from 3 to 100 ng/ml produced a dose-dependent relaxation in the lymphatic preparations precontracted by 10(-7) M bradykinin. The ANP-induced relaxation was not modified by pretreatment with 5 x 10(-7) M propranolol, 5 x 10(-7) M atropine, 10(-6) M cimetidine, 5 x 10(-5) M aspirin, or 10(-5) M ouabain. The mechanical rubbing of endothelial cells in the lymph vessels caused no significant effect on the ANP-induced relaxation. The relaxation, however, was significantly reduced by pretreatment with 10(-5) M methylene blue. These results suggest that ANP in a low concentration seems to inhibit lymph transport through a reduction of spontaneous contractions and a marked relaxation of lymphatic smooth muscles in bovine mesenteric lymphatics and that ANP may produce the relaxation through synthesis of guanosine 3',5' cyclic monophosphate, independent of the lymphatic endothelium.