Ecdysteroid content in ovaries of Bombyx larvae was determined every 12 h through the feeding period in the fifth instar. The content was less than 5 pg/ovary pair on day 0 to day 2, but more than 20 pg/ovary pair on day 3. It then returned to the original level 12 h later and remained at a low level until day 6. This increase in the ecdysteroid content found on day 3 was entirely suppressed by an application of juvenile hormone analogue. Cultured ovaries did not release ecdysteroids into the culture medium, and neither crude brain extract nor PTTH elevated ecdysteroid content in ovaries isolated from day 2 larvae. Ecdysteroids in the ovaries of day 3 larvae were tentatively identified as ecdysone, 3-dehydroecdysone, 20-hydroxyecdysone and other ecdysteroids by the combination of high performance liquid chromatography and radioimmunoassay. Growth of the ovaries in the fifth instar commenced on day 4. Ecdysteroids found on day 3, when the haemolymph ecdysteroid titre remains at a low level, may trigger further development of ovaries in the fifth instar.