Cephalosporin-C (CPC) deacetylase [EC 184.108.40.206] (CAH; systemic name, cephalosporin-C acetylhy-drolase) plays a role in deacetylation of C-3 of CPC antibiotics. In this study we obtained the various lipase secreting microorganism by using a tributyrin agar plate method. Visible clear zones due to lipase microorganism, Pseudomonas, having the activity of CPC deacetylase by means of thin layer chromatography (TLC). Supernatants of candidate bacteria culture were reacted with cephalosporin-C and then spotted cellulose plate and was developed with mixture of isopropanol and water (ratio 7 to 3). The CPC deacetylase gene was cloned in E.coli JM109 and sequenced. The cloned DNA(pUCLip) was 2.6kb in length. The nucleotide sequence revealed the presence of one major open reading frame of 1905 nucleotides and encoded a polypeptide consisting of 634 amino acids. The initiation codon of cephalosporin-C deacetylase gene was ATG. The G+C content of the enzyme was 62%. The deduced amino acid sequence showed 58% identity to EstA of Pseuomonas aeruginosa. The consensus sequence maintained among other microorganism cephalosporin-C deacetylases, Gly-X-Ser-X-Gly, was not observed in this protein. Instead cephalosporin-C deacetylase contained the common sequence Gly-Asp-Ser-Leu found in the GDSL family of lipolytic enzymes.