D. melanogaster CK2 (DmCK2) is a highly conserved protein kinase that is composed of catalytic, alpha, and regulatory, beta, subunits associated as an alpha2beta2 heterotetramer. In order to analyze the functions of CK2 in this metazoan model, we have used the two hybrid approach to identify interacting proteins. One of these cDNAs, DmA24, encodes a novel polypeptide with no homologs in GenBank, and is notable in that it contains a bipartite nuclear localization signal and two sites for phosphorylation by CK2. In situ hybridization to polytene chromosomes indicates that the DmA24 gene is located at the 61 D interval of chromosome II a region that also harbors 3 additional genes with similar structure. DmA24p interacts with DmCK2alpha, but not with DmCK2beta, demonstrating that this interaction is specific for the catalytic subunit of CK2. In addition, the protein is phosphorylated by the holoenzyme purified from Drosophila embryos. These studies identify DmA24p as a potentially new physiological partner of DmCK2. In addition, we also report the results of a large-scale screen that has identified a new set of DmCK2-interacting proteins. Most notable among these are Surf6, a nucleolar protein involved in RNA processing, and Spalt, a homeotic protein.