Hemolin is the only insect member of the immunoglobulin (Ig) superfamily reported to be up-regulated during an immune response. In diapausing pupae of Hyalophora cecropia the gene is expressed in fat body cells and in haemocytes. Like the mammalian Ig kappa light chain gene, the Hemolin gene harbours an enhancer including a kappaB motif in one of its introns. This motif binds the H. cecropia Rel factor Cif (Cecropia immunoresponsive factor). The Hemolin third intron also mediates transient reporter gene expression in immunoresponsive Drosophila mbn-2 cells. Co-transfections of Drosophila SL2 cells showed that the Drosophila Rel factor Dif (Dorsal-related immunity factor), transactivates reporter gene constructs through the intron. Moreover, a 4.8-fold synergistic activation was obtained when Dif is combined with the rat C/EBP (CCAAT/enhancer element-binding protein) and human HMGI (high mobility group protein I). This is the first report of an insect immune-related gene that is up-regulated by an enhancer activity conferred through an intron.