The IAL-PID2 cells derived from imaginal wing discs of the last larval instar of Plodia interpunctella were responsive to 20-hydroxyecdysone (20E). These imaginal cells respond to 20E by proliferative arrest followed by a morphological differentiation. These 20E-induced late responses were inhibited in presence of juvenile hormone (JH II). From these imaginal wing cells, we have cloned a cDNA sequence encoding a P. interpunctella ecdysone receptor-B1 isoform (PIEcR-B1). The amino acid sequence of PIEcR-B1 showed a high degree of identity with EcR-B1 isoforms of Bombyx mori, Manduca sexta and Choristoneura fumiferana. The pattern of PIEcR-B1mRNA induction by 20E was characterized by a biphasic response with peaks at 2 h and 18 h. The presence of the protein synthesis inhibitor anisomycin induced a slight reduction in level of PIEcR-B1 mRNA and prevented the subsequent declines observed in 20E-treated cells. Therefore, PIEcR-B1 mRNA was directly induced by 20E and its downregulation depended on protein synthesis. An exposure of imaginal wing cells to 20E in the presence of JH II caused an increased expression of Plodia E75-B and HR3 transcription factors but inhibited the second increase of PIEcR-B1 mRNA. These findings showed that in vitro JH II was able to prevent the 20E-induced differentiation of imaginal wing cells. This effect could result from a JH II action on the 20E-induced genetic cascade through a modulation of EcR-B1, E75-B and HR3 expression.