Rabbit pancreatic cholesterol esterase (CEase, carboxyl ester lipase, EC 3.1.1.3) has been cloned from a lambda gt11 library of adult rabbit pancreatic cDNA. The open reading frame consists of 1788 nucleotides which encodes 576 amino acids of the functional protein and a 20 amino acid leader peptide. When compared to other species, the greatest homology is observed between residues 82-248 with little or no homology at the C-terminal end where proline-glutamate-serine-threonine (PEST) segments are a characteristic feature of the human CEase. Rabbit CEase (RCEase) retains the active-site serine (gxsxg), the active-site histidine and the tentative heparin binding site (KKRCLQ) at similar positions in comparison to pancreatic CEases of other species. When rabbit CEase cDNA is expressed in monkey kidney (COS-7) cells, enzymatic hydrolytic activity is detected in the growth medium as is a 67 kDa protein by Western blotting with polyclonal anti-CEase antibody. Northern blot analysis shows two mRNA (2.2 and 3.2 kb) species.