2[125I]Iodomelatonin binding sites were identified in the mucosa of the human colon from Chinese patients with carcinoma of the rectum or colon using biochemical receptor assay and autoradiography. Melatonin in the colonic mucosa/submucosa and muscle layers were quantitated by radioimmunoassay. The binding of 2[125I]iodomelatonin to the membrane preparations of the human colonic mucosa/submucosa was stable, saturable, reversible and of high affinity. Rosenthal analysis from saturation studies performed at 21 degrees C yielded an equilibrium dissociation constant (Kd) of 61.7 +/- 4.48 pmol/L (n = 3) and maximum number of binding sites (B(max)) of 1.65 +/- 0.51 fmol/mg protein (n = 3). The linearity of the Rosenthal plots and unity of the Hill coefficient suggested that 2[125I]iodomelatonin was bound to a single class of binding sites. The radioligand binding was displaced by 2-iodomelatonin (Ki = 0.02 nmol/L), melatonin (0.65 nmol/L), 6-chloromelatonin (Ki = 5.33 nmol/L), 6-hydroxymelatonin (Ki = 33.8 nmol/L) and N-acetylserotonin (Ki = 122 nmol/L). The characteristic of the binding sites were similar to those reported in the jejunum of duck, chicken, and human but of higher affinity than those in the mouse colon. Autoradiography localizes the binding to the mucosa of the human colon. Radioimmunoassay revealed a melatonin concentration of 467 +/- 99 pg/g wet tissue of human colon (n = 6). Our findings suggest that melatonin may influence the human colonic functions through interaction with its receptors in the mucosa.