A neutral ceramidase activity has earlier been identified in the intestinal mucosa and gut lumen and postulated to be important in the digestion of sphingolipids. It is found throughout the intestine, but has never been fully characterized. We now purified rat intestinal neutral ceramidase from an eluate obtained by perfusing the intestinal lumen with 0.9 % NaCl and 3mM sodium taurodeoxycholate. Using a combination of acetone precipitation, ion exchange, hydrophobic interaction and gel chromatographies we obtained a homogenous enzyme protein with a molecular mass around 116 kDa. The enzyme acts on both ¹⁴C-octanoyl- and ¹⁴C-palmitoyl sphingosine in the presence of glycocholic and taurocholic acid and the bile salt analogue 3-[(3-Cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS), but inhibited by 2 mM or more of other bile salts. It is a glycosylated protein that is stable to trypsin and chymotrypsin exposure is not influenced by Ca²⁺, Mg²⁺ or Mn²⁺ ions and is inhibited by Zn²⁺ and Cu²⁺ ions. Mass fragmentographic analysis (MALDI-TOF) identified twelve fragments covering 17.5 % of the sequence for neutral/alkaline ceramidase 2 purified by Mitsutake et al (J Biol Chem 2001; 276:26249-59) from rat kidney and found to be located in the apical membrane of renal tubular cells. The intestinal and the kidney ceramidases also have similar molecular mass and ion dependence. The intestinal ceramidase thus is a neutral ceramidase 2 that is released by bile salts and is resistant to pancreatic proteases. It is well suited to metabolize ceramide formed from dietary and brush border sphingolipids to generate other bioactive sphingolipid messengers.