Ecdysteroid binding was investigated in Kc cells and larval fat body of Drosophila. After incubation of intact Kc cells, 200-300 specific binding sites were detected for both 20-OH ecdysone and ponasterone A. However, if cells were fractionated prior to incubation with 20-OH ecdysone, no specific binding was observed. In the fat body the number of specific nuclear binding sites appears to be enhanced by the factor of polyteny if compared with Kc cells. Both systems displayed a high degree of non-specific cytoplasmic binding. The intracellular distribution of specifically bound ponasterone A was different from that of the physiological hormones. In fat body, even after an incubation of 60 min at 22 degrees C, the majority of specifically bound ponasterone A was located in the cytoplasm. Differences between the results of this study and previous reports on ecdysteroid binding are discussed.