Two novel esterases (EstB1 and EstB2) were isolated from a genomic library of Bacillus sp. associated with the marine sponge Aplysina aerophoba. EstB1 shows low identity ( 26 - 44%) with the published hydrolases of the genus Bacillus, whereas EstB2 shows high identity ( 73 - 74%) with the carboxylesterases from B. cereus and B. anthracis. Both esterases were efficiently expressed in Escherichia coli under the control of T7 promoter using the vector pET-22b(+). Recombinant EstB1 was purified in a single step to electrophoretic homogeneity by IMAC. A method for the refolding of inclusion bodies formed by the recombinant EstB2 was established to obtain active enzyme. Substrate specificity of the two enzymes towards p-nitrophenyl and methyl esters and the respective kinetic parameters K-m and V-max were determined. The temperature optima of EstB1 and EstB2 were determined to be in the range of 30 - 50 degrees C and 20 - 35 degrees C, respectively. The pH optima were found to be in the range of 6.5 - 7.5 and 6.5 - 8.0, respectively. Both enzymes showed the highest stability in up to 50% (v/v) DMSO followed by methanol, ethanol and 2-propanol. The influence of high NaCl and KCl concentrations was tested. The inhibition effect of 10 50 mM Zn2+ and 50 mM Mg2+ and Ca2+ ions was observed for both esterases. One to five millimolar PMSF deactivated the enzymes, whereas beta-mercaptoethanol, DTT and EDTA had no effect on the enzymes activity.