A blood meal induces the ovaries of female Aedes aegypti mosquitoes to produce ecdysteroid hormones that regulate many processes required for egg maturation. Various proteins involved in the intracellular transport and biosynthesis of ecdysteroid precursors have been identified by analysis of Drosophila melanogaster mutants and by biochemical and molecular techniques in other insects. To begin examining these processes in mosquito ovaries, complete cDNAs were cloned for putative orthologs of diazepam-binding inhibitor (DBI), StAR-related lipid transfer domain containing protein (Start1), aldo/keto reductase (A/KR), adrenodoxin reductase (AR), and the cytochrome P450 enzymes, CYP302a1 (22-hydroxylase), CYP315a1 (2-hydroxylase) and CYP314a1 (20-hydroxylase). As shown by RT-PCR, transcripts for all seven genes were present in ovaries and other tissues both before and following a blood meal. Expression of these genes likely supports the low level of ecdysteroids produced in vitro (7-10 pg /tissue/6 h) by tissues other than ovaries. Ovaries from females not blood fed and up to 6 h post blood meal (PBM) also produced low amounts of ecdysteroids in vitro, but by 18 and 30 h PBM, ecdysteroid production was greatly increased (75-106 pg/ovary pair/6h) and thereafter (48 and 72 h PBM) returned to low levels. As determined by real-time PCR analysis, gene transcript abundance for AedaeCYP302 and AedaeCYP315a1 was significantly greater (9 and 12 fold, respectively) in ovaries during peak ecdysteroid production relative to that in ovaries from females not blood fed or 2 h PBM. AedaeStart1, AedaeA/KR and AedaeAR also had high transcript levels in ovaries during peak ecdysteroid production, and AedaeDBI transcripts had the greatest increase at 48 h PBM. In contrast, gene transcript abundance of AedaeCYP314a1 decreased PBM. This study shows for the first time that transcription of a few key genes for proteins involved in ecdysteroid biosynthesis is positively correlated with the rise in ecdysteroid production by ovaries of a female insect.