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An elevated concentration in the colon of the primary bile acid chenodeoxycholic acid (CDCA) or the secondary bile acid deoxycholic acid (DCA) is known to induce water secretion, causing diarrhea. We hypothesized that of the many fecal bile acids, only CDCA and DCA function as endogenous laxatives; therefore, a decrease in their proportion may be a cause of childhood functional constipation. To test this possibility, fecal bile acid composition was determined in children with functional constipation and in nonconstipated control children.Fecal samples were obtained from 207 children, 103 with functional constipation and 104 with normal bowel habits. Bile acid classes were determined by use of electrospray ionization—single ion monitoring—mass spectrometry (ESI-SIM-MS), and individual bile acids were measured by gas chromatography (GC)-MS (GC-MS). The structure of individual sulfated bile acids was obtained by use of liquid chromatography (LC)-MS (LC-MS).By ESI-SIM-MS, the proportions of DCA did not differ in constipated children (n = 73) from that in control children (n = 92), but monosulfated dihydroxy bile acids were greater (P < 0.05). The difference was attributable to 6 patients in the constipated group whose major fecal bile acid by LC-MS was the 3-sulfate of CDCA. Sulfation of CDCA is known to abolish its secretory activity. By GC-MS, the bile acid profile was identical in the 2 groups.In most children with functional constipation, the fecal bile acid profile seems to be normal. There is a small subset of children, however, whose dominant fecal bile acid is the 3-sulfate of CDCA, indicating a novel disturbance in bile acid metabolism. Such sulfation abolishes the secretory activity of CDCA and may contribute to constipation.