Ankyrins are linker proteins, which connect various membrane proteins, including members of the L1 family of neural cell adhesion molecules, with the submembranous actin–spectrin skeleton. Here we report the cloning and characterization of a second, novelDrosophilaankyrin gene (Dank2) that appears to be the result of a gene duplication event during arthropod evolution. TheDrosophilaL1-type protein neuroglian interacts with products from bothDrosophilaankyrin genes. Whereas the previously described ankyrin gene is ubiquitously expressed during embryogenesis, the expression ofDank2is restricted to the nervous system in theDrosophilaembryo. The absence of neuroglian protein in a neuroglian null mutant line causes decreased levels of Dank2 protein in most neuronal cells. This suggests that neuroglian is important for the stability of Dank2 protein. However, neuroglian is not required for Dank2 axonal localization. In temperature-sensitiveneuroglianmutants in which neuroglian protein is mislocated at the restrictive temperature to an intracellular location in the neuronal soma, Dank2 protein can still be detected along embryonic nerve tracts.