In the silkworm, Bombyx mori, it has been demonstrated that most free ecdysteroids in the ovary are converted to physiologically inactive ecdysteroid 22-phosphates, which are then transformed back to free ecdysteroids during early embryonic development. Two specific enzymes involved in the reciprocal conversion of ecdysteroids, namely, ecdysteroid 22-kinase (EcKinase) and ecdysteroid-phosphate phosphatase, have been isolated and characterized. In this study, we first attempted a phylogenetic analysis of EcKinase. The resulting phylogenetic tree showed that many proteins homologous to B. mori EcKinase are found not only in ecdysozoa, including insects and nematodes, but also in teleosts, fungi, and bacteria. We then investigated the sites where free ecdysteroids are synthesized and phosphorylated in the ovary. We found that (1) the mRNAs of two P450 enzymes involved in ecdysteroidogenesis, CYP306a1 (25-hydroxylase) and CYP314a1 (20-hydroxylase), are expressed mainly in follicle cells, (2) EcKinase mRNA localizes in the oocyte and nurse cells, and (3) EcKinase immunoreactivity localizes mainly in the external region of the oocyte, not in nurse cells or follicle cells. From these results, we suggest that ecdysteroids in the B. mori ovary are synthesized in follicle cells and transferred into the oocyte, where they are phosphorylated by EcKinase, whose mRNA originates from nurse cells and the oocyte itself.