We have isolated two Drosophila lines that carry point mutations in the gene coding for the NF-KB-like factor DIF. Like mutants of the Toll pathway, Dif mutant flies are susceptible to fungal but not to bacterial infections. Genetic epistasis experiments demonstrate that Dif mediates the Toll-dependent control of the inducibility of the antifungal peptide gene Drosomycin. Strikingly, DIF alone is required for the antifungal response in adults, but is redundant in larvae with Dorsal, another Rel family member. In Drosophila, Dif appears to be dedicated to the antifungal defense elicited by fungi and gram-positive bacteria. We discuss in this light the possibility that NF-KB1/p50 might be required more specifically in the innate immune response against gram-positive bacteria in mammals.