The diazepam-binding inhibitor (DBI) is a 10-kDa highly evolutionarily conserved multifunctional protein. In mammals, one of DBI's functions is in the activation of steroid hormone biosynthesis via binding to a specific outer mitochondrial membrane receptor (benzodiazepine receptor, BZD) and promoting cholesterol transport to the inner membrane. In this work, a multitiered approach was utilized to study the role of this receptor-like activity in ecdysteroidogenesis by larval insect prothoracic glands (PGs). First, both DBI protein and messenger RNA (mRNA) levels were correlated with peak PG ecdysteroid production. In vitro ecdysteroid production was stimulated by the diazepam analogue FGIN 1-27 and inhibited anti-DBI antibodies. The DBI protein was found distributed throughout PG cells, including regions of dense mitochondria, supposed subcellular sites of ecdysteroid synthesis. Finally, a potential mitochondrial BZD receptor in PG cells was demonstrated by photoaffinity labeling. These results suggest an important role for the insect DBI in the stimulation of steroidogenesis by prothoracic glands and indicate that a pathway for cholesterol mobilization leading to the production of steroid hormones appears to be conserved between arthropods and mammals.