Abstract A putative esterase gene ( est) from Acinetobacter calcoaceticus RAG-1 has been cloned into Escherichia coli. Esterase-positive clones exhibited high levels of esterase activity even in intact cells. In addition, expression of the est gene conferred on E. coli the ability to grow on simple triglycerides such as triacetin (TAC). The original esterase-positive plasmid pRA17 carried a 2.2-kb insert from a partial MboI digest of RAG-1 DNA, which gave a single band with RAG-1 DNA following Southern hybridization. By subcloning and sequencing the est gene was found to contain a sequence of 870 bp which could be translated to yield a protein of M r 32700. In support of the sequencing results was the finding that when pRA17 was expressed in minicells, a unique peptide of M r 32500 was identified. This peptide was not found in minicells transformed with esterase-negative plasmids, such as pRA176, which contained a Tn 5 insertion in the est gene. The fact that the production of active esterase depended on the orientation of the est gene within the vector suggested that transcription proceeded from the tet promoter in pBR322.