Signaling by Drosophila Toll pathway activates two Rel/NF-kappaB transcription factors, Dorsal (Dl) and Dorsal-related immune factor (Dif). Dl plays a central role in the establishment of dorsoventral polarity during early embryogenesis, whereas Dif mediates the Toll receptor-dependent antifungal immune response in adult Drosophila. The absence of a Dif ortholog in mosquito genomes suggests that Dl may play its functional role in the mosquito Toll-mediated innate immune responses. We have cloned and molecularly characterized the gene homologous to Drosophila Dl and to Anopheles gambiae REL1 (Gambif1) from the yellow fever mosquito Aedes aegypti, named AaREL1. AaREL1 alternative transcripts encode two isoforms, AaREL1-A and AaREL1-B. Both transcripts are enriched during embryogenesis and are inducible by septic injury in larval and female mosquitoes. AaREL1 and AaREL2 (Aedes Relish) selectively bind to different kappaB motifs from insect immune gene promoters. Ectopic expression of AaREL1-A in both Drosophila mbn-2 cells and transgenic flies specifically activates Drosomycin and results in increased resistance against the fungus Beauveria bassiana. AaREL1-B acted cooperatively with AaREL1-A to enhance the immune gene activation in Aag-2 cells. The RNA interference knock-outs revealed that AaREL1 affected the expression of Aedes homologue of Drosophila Serpin-27A and mediated specific antifungal immune response against B. bassiana. These results indicate that the homologue of Dl, but not that of Dif, is a key regulator of the Toll antifungal immune pathway in A. aegypti female mosquitoes.