In the present study, we reveal for the first time that the Bactrocera oleae chorion peroxidase (bPxd) participates essentially in B. oleae chorion formation and clearly represents the homologous member of Drosophila melanogaster chorion peroxidase (Pxd). Comparative sequence analysis disclosed that the bPxd cDNA semi-central region, which encodes for the putative catalytic domain of the enzyme, exhibits great homology (98%) with its Pxd counterpart. Thus, it is very likely that bPxd is highly responsible for the chorion hardening process, through protein cross-linking mediated by the formation of di- and tri-tyrosine bonds. Distinct molecular weight bPxd RNA transcripts were detected in Northern blotting analysis of total RNA extracts of adult flies (2.9 and 1.7 kb) and ovaries (2.2 kb). The ovarian-specific bPxd RNA transcript is selectively expressed in the follicle cell layer during the late stages of oogenesis 12-14, as revealed by in situ hybridization. Moreover, reverse transcription reactions confirmed the stage-specific developmental regulation of the bPxd gene, which is maximally expressed during stage 13. Western blotting with the rabbit anti-rAePO polyclonal antibody revealed three immunoreactive bands of 76, 66 and 54 kDa in crude protein extracts from adult flies, while in larva and purified chorion preparations, a unique 54 kDa band was clearly detected. Immunolocalization experiments revealed that bPxd peroxidase constitutes an essential structural chorionic component, being abundantly localized in all the successive chorionic layers and vitelline membrane as well.