A putative juvenile hormone esterase (JHE) binding protein, P29, was isolated from the tobacco hornworm Manduca sexta [J. Biol. Chem. 275(3), 1802-1806]. A homolog of P29 was identified in Drosophila melanogaster by sequence alignment. This gene, CG3776 was cloned, recombinant DmP29 expressed in Escheriscia coli and two anti-DmP29 antisera raised. In vitro binding of the P29 homolog to Drosophila JHE was confirmed. P29 mRNA and an immunoreactive protein of 25 kDa were detected in Drosophila larvae, pupae and adults. The predicted size of the protein is 30 kDa. Drosophila P29 is predicted to localize to mitochondria (MitoProt; 93% probability) and has a 6 kDa N-terminal targeting sequence. Subcellular organelle fractionation and confocal microscopy of Drosophila S2 cells confirmed that the immunoreactive 25 kDa protein is present in mitochondria but not in the cytosol. Expression of P29 without the predicted N-terminal targeting sequence in High Five cells showed that the N-terminal targeting sequence is shorter than predicted, and that a second, internal mitochondrial targeting signal is also present. An immunoreactive protein of 50 kDa in the hemolymph does not result from alternative splicing of CG3776 but may result from dimerization of P29. The function of P29 in mitochondria and the possible interaction with JHE are discussed.