Ecdysteroid localization in target tissues ofDrosophila melanogasterwas demonstrated by the utilization of low temperature autoradiography and immunohistochemistry. Autoradiographic studies utilized high specific activity [3H]ponasterone A, an ecdysteroid with exceptional molting hormone activity inDrosophila.The data revealed a selective nuclear concentration of the labelled ligand in cells of the salivary gland, corpus allatum (source of juvenile hormone), prothoracic gland (source of ecdysone) and the central nervous system. Nuclear localization was also observed in epithelial cells of the esophagus and muscle cells of the pharynx and mandibles, etc. The specific labelling of cell nuclei was prevented by incubation of the tissue with [3H]ponasterone A in the presence of excess unlabelled ponasterone A. Nuclear localization of ponasterone A in the salivary gland was also observed when using an antiserum against ecdysteroids and an avidin-biotin-complex (ABC) detection system. The staining showed two binding patterns, heavy and light, that agreed with the autoradiographic studies. The results clearly demonstrate specific ponasterone A binding sites in nuclei of the target cells and support the dogma that ecdysteroids exert their effects at the level of the genome.