Genital imaginal discs were dissected fromHeliothis virescenspupae just after diapause break and culturedin vitro.The discs continued to grow and differentiate when maintained in media containing sufficiently high titers of 20-hydroxyecdysone (20HE) and co-cultured fat body or testis sheath tissue, or extract of fat body previously exposed to 20HEin vitro.Continuous changes were observed for approximately 30 days. Other tissues provided only temporary (5–8 day) developmental support. Genetal discs did not develop in the absence of 20 HE. Discs co-cultured with fat body in 20 HE medium synthesized more protein and DNA than discs co-cultured with fat body but without 20 HE. Fat body and testis sheath tissue, cultured with sufficient titers of 20 HE, produced factors essential to the development of genital imaginal discs ofH. virescens.