Hange-shashin-to has been used for chronic hypofunction of the gastrointestinal tract and to improve functional abnormalities of the upper and lower gastrointestinal system. To determine whether the pharmacological effects of Hange-shashin-to are due to gut-regulatory peptide levels, we developed a sensitive and specific double-antibody enzyme immunoassay (EIA) for detecting motilin and also examined the levels of somatostatin-, motilin-, gastrin-, and vasoactive intestinal peptide (VIP)-immunoreactive substances (IS) in plasma from healthy subjects. We developed a sensitive (3.5 pg, 1.4 pg/well) and specific (carboxy-terminal region) EIA for motilin. A single oral administration of Hange-shashin-to 6.0 g caused significant increases somatostatin-IS (20-60 min), motilin-IS (40 min), and gastrin-IS (40-90 min) levels in plasma compared with levels in a placebo group. Hange-shashin-to had no significant effect on VIP-IS levels after single administration. These changes in hormone levels (somatostatin, motilin, and gastrin) might relate to normalization of the upper and lower gastrointestinal system by Hange-shashin-to.