Juvenile hormone esterase (JHE) plays an important role in regulating juvenile hormone titers during insect life cycle. Recent sequencing and annotation of Aedes aegypti genome identified 10 putative JHE gene sequences. We performed further analysis on these 10 putative JHE gene sequences and found that only two of them identified with NCBI accession numbers, EAT43357 and EAT43353, contained most of the conserved catalytic motifs identified in JHE genes cloned from other species of insects. To determine if either of these two putative JHE genes code for functional JHE, we have measured the mRNA profiles of these two genes during last larval and pupal stages by using Real-time quantitative RT-PCR. EAT43353 mRNA levels were higher at the early final instar larval stage and the levels decreased towards the end of this stage. In contrast, EAT43357 mRNA levels were lower at the beginning of final instar larval stage and increased with age reaching the maximum levels by the end of larval stage, 42-45 hr after ecdysis into the final instar larval stage (AEFL). EAT43357 mRNA was expressed in both midgut and fat body tissues. Partition assay was used to monitor the enzymatic activity of JHE. The JHE enzyme levels gradually increased during final instar larval stage and reached the peak at 42 hr AEFL. Complete open reading frame of EAT43357 was expressed in a baculovirus system and produced a protein that showed JHE enzymatic activity.