Using cDNAs for the human retinoic acid receptor alpha (hRAR alpha) and Drosophila hormone receptor 3 (DHR3), we isolated a cDNA encoding a member of the steroid hormone receptor superfamily from the tobacco hornworm, Manduca sexta. Sequencing showed that this cDNA is most closely related to DHR3 (97 and 68% amino acid identity in the DNA and ligand binding regions, respectively) followed by hRAR alpha (65 and 20% identity, respectively) and therefore is named MHR3. The cDNA hybridized to two mRNAs (3.8 and 4.5 kb) found in the epidermis during the ecdysteroid rises for the embryonic, larval, and pupal molts. Culture of fourth instar larval epidermis with 4 microM 20-hydroxyecdysone (2 micrograms/ml 20HE) caused the appearance of MHR3 mRNA within 3 hr and maximal expression by 6 hr; after 12 hr continuous exposure to 20HE, the mRNA level declined. The 4.5-kb mRNA appeared first, both were present in equal amounts by 12 hr, and by 20 hr the predominant transcript was 3.8 kb. Similar 20HE-induced expression was seen in epidermis explanted 1 day after the onset of wandering, although with a slower time course. The induction was largely independent of protein synthesis, but the subsequent decline required protein synthesis as is typical of the "early" puffs in Drosophila. Continuous exposure to 20HE was necessary for MHR3 expression; in its absence, the mRNA declined with a half-life of 2 hr. Thus, MHR3 is an ecdysteroid-inducible DNA binding protein that likely is a transcription factor involved in the cascade of gene activation and inactivation caused by ecdysteroids during the insect molt.