Some refractory anopheline mosquitoes are capable of killing Plasmodium, the causative agent of malaria, by melanotic encapsulation of invading ookinetes. Phenoloxidase (PO) appears to be involved in the formation of melanin and toxic metabolites in the surrounding capsule. A cDNA encoding Anopheles stephensi prophenoloxidase (Ans-proPO) was isolated from a cDNA library screened with an amplimer produced by reverse transcriptase polymerase chain reaction (RT-PCR) with degenerate primers designed against conserved proPO sequences. The 2.4-kb-long cDNA has a 2058 bp open reading frame encoding Ans-proPO of 686 amino acids. The deduced amino acid sequence shows significant homology to other insect proPO sequences especially at the two putative copper-binding domains. In A. stephensi, Ans-proPO expression was detected in larval, pupal and adult stages. The Ans-proPO mRNA was detected by RT-PCR and in situ hybridization in haemocytes, fat body and epidermis of adult female mosquitoes. A low level of expression was detected in the ovaries, whereas no expression was detected in the midguts. Semi-quantitative RT-PCR analysis of Ans-proPO mRNA showed that its expression was similar in adult female heads, thoraxes and abdomens. No change in the level of Ans-proPO expression was found in adult females after blood feeding, bacterial challenge or Plasmodium berghei infection. However, elevated PO activity was detected in P. berghei-infected mosquitoes, suggesting that in non-selected permissive mosquitoes PO may be involved in limiting parasite infection. Genomic Southern blot and immunoblots suggest the presence of more than one proPO gene in the A. stephensi genome, which is consistent with the findings in other Diptera and Lepidoptera species. The greatest similarity in sequence and expression profile between Ans-proPO and A. gambiae proPO6 suggests that they might be homologues. Our results demonstrate that Ans-proPO is constitutively expressed through different developmental stages and under different physiological conditions, implying that other factors in the proPO activation cascade regulate melanotic encapsulation.