We have investigated the molecular and cellular mechanisms involved in the control of insect cell cycle by 20-hydroxyecdysone (20E) using the IAL-PID2 cell line established from imaginal wing discs of Plodia interpunctella. We first defined conditions for use of hydroxyurea, a reversible inhibitor of DNA synthesis, in order to synchronize the IAL-PID2 cells in their division cycle. A high degree of synchrony was reached when cells were exposed to two consecutive hydroxyurea treatments at 1 mm for 36 h spaced 16 h apart. Under these conditions, flow cytometry analysis demonstrated that 20E at 10(-6) m induced an inhibition of cell growth by an arrest of 90% of the cells in G2/M phase. Using cDNA probes specifically designed from E75 and HR3 nuclear receptors of Plodia interpunctella, we showed that PiE75 and PHR3 were highly induced by 20E through S and G2 phases with maximal enhancement just before the G2/M arrest of cells. These findings suggest that PiE75 and PHR3 could be involved in a 20E-induced genetic cascade leading to G2/M arrest.