We report the cloning and characterization of two isoforms of the Ultraspiracle homologue (AaUSP) from the mosquito, Aedes aegypti. The 2.33-kb AaUSPa cDNA has an open reading frame (ORF) of 484 amino acids encoding a polypeptide of 54 kDa, whereas the 2.14-kb AaUSPb ORF of 459 amino acids encodes a 51.3 kDa polypeptide. The AaUSPa and AaUSPb proteins differ only in the N-terminal portion of the variable A/B domain. The AaUSP DNA-binding domain shares 92% and 97% identities with the respective domains of the Drosophila (DmUSP) and Bombyx (BmUSP) Ultraspiracles. However, the AaUSP ligand-binding domain is only 57% and 52% identical to those of DmUSP and BmUSP, respectively. In spite of the relatively low level of sequence conservation, electrophoretic mobility shift assay (EMSA) and hormone-binding assay clearly demonstrated that the products of the AaUSPa and AaUSPb cDNAs are functional heterodimeric partners of the mosquito ecdysteroid receptor. In vitellogenic tissues, each of the two AaUSP isoforms is expressed differently: the AaUSPa is predominant in the fat body and the AaUSPb in the ovary. The kinetics of ovarian AaUSP mRNA coincide with those of the ecdysteroid receptor, being elevated during the previtellogenic period and shortly after the onset of vitellogenesis. In contrast, the level of the AaUSP in the fat body remains relatively constant throughout most of the vitellogenic cycle.