The esterase-encoding gene, estA, was cloned from Acinetobacter lwoffii 16C-1 genomic DNA into Escherichia coli BL21(DE3) with plasmid vector pET-22b (pEM1). pEM1 has a 4.4-kb EcoRI insert that contained the complete estA gene. A 2.4-kb Aval-SphI DNA fragment was subcloned (pEM3) and sequenced. estA gene encodes a protein of 366 amino acids (40,687 Da) with a pI of 9.17. The EstA signal peptide was 31 amino acids long, and the mature esterase sequence is 335 amino acids long (37.5 kDa). The conserved catalytic serine residue of EstA is in position 210. The EstA- sequence was similar to that of the carboxylesterase from Acinetobacter calcoaceticus (75% identity, 85% similarity), Archaeoglobusfulgidus (37% identity, 59% similarity), and Mycobacterium tuberculosis (35% identity, 51% similarity). These enzymes contained the conserved motif G-X-1-S-X-2-G carrying the active-site serine of hydrolytic enzyme. The EstA activity in A. lwoffii 16C-1 remains constant throughout the stationary phase, and the activity in E. coil BL21 (DE3) with pEM1 was similar to A. lwoffii 16C-1.