To obtain information on the concentration and spectrum of bile acids in human cecal content, samples were obtained from persons dying an unnatural death, such as caused by trauma, homicide, suicide, or drug overdose. Bile acid concentration was measured using an enzymatic assay for 3-hydroxy bile acids; bile acid classes were determined by electrospray ionization mass spectrometry (ESI-MS); and individual bile acids by gas-chromatography-mass spectrometry (GC-MS) and liquid chromatography (LC)-MS. The 3-hydroxy bile acid concentration (µmol bile acid/ml cecal content) was 0.4 ± 0.2 mM (mean ± SD); the total 3-hydroxy bile acid concentration was 0.6 ± 0.3 mM. The aqueous concentration of bile acids (supernatant after centrifugation) was identical, indicating that most bile acids were in solution. By LC-MS, bile acids were mostly in unconjugated form (90 ± 9%, mean ± SD); sulfated, non-amidated bile acids were 7 ± 5%, and non-sulfated amidated bile acids (glycine or taurine conjugates), were 3 ± 7%. By GC-MS, ten bile acids were identified: deoxycholic (34 ± 16%), lithocholic (26 ± 10%), ursodeoxycholic, (6±9) as well as their primary bile acid precursors cholic (6 ± 9%) and chenodeoxycholic acid (7 ± 8%). In addition, 3-hydroxy derivatives of some or all of these bile acids were present and averaged 27 ± 18% of total bile acids, indicating that 3-hydroxy bile acids are normal constituents of cecal content. In the human cecum, deconjugation and dehydroxylation of bile acids are nearly complete, resulting in most bile acids being in unconjugated form at submicellar and subsecretory concentrations.