Aims In this study, a gene that encodes a carboxylesterase (carb) in Penicillium expansum GF was cloned, sequenced and overexpressed by Penicillium griseoroseum PG63, and the enzyme was characterized. Methods and Results The recombinant strain, P.griseoroseum T55, obtained upon transformation using the plasmid pAN-52-1-carb, showed integration of the carb gene into at least two heterologous sites of the genome by Southern blotting. Furthermore, the recombinant strain T55 exhibited almost a fourfold increase in carboxylesterase activity compared with PG63 strain when both were cultured without inducers. Based on the secondary structure and multiple sequence alignments with carboxylesterases, cholinesterase and lipase, a three-dimensional model was obtained. The / barrel topology, that is typical of esterases and lipases, was indicated for the CARB protein with Ser213-Glu341-His456 as the putative catalytic triad. CARB preferentially hydrolysed acyl chains with eight carbon atoms, and its activity was optimal at a pH of 7 center dot 0 and a temperature of 25 degrees C. CARB exhibited stability in alkaline pH, high activity under mesophilic conditions and stability in organic solvents. Conclusion The CARB protein is potentially useful in bioremediation, food and chemical/pharmaceutical industries. Significance and Impact of the Study This study is the first to report the development of a recombinant strain superproducing a Penicillium sp. carboxylesterase.