Drosophila melanogaster casein kinase II (CKII) is composed of catalytic alpha and regulatory beta subunits that generate the alpha(2)beta(2) holoenzyme. A two-hybrid screen of a Drosophila embryo library using CKIIalpha as bait has resulted in the isolation of multiple cDNAs encoding SSL, a CKIIbeta-like polypeptide. We demonstrate that CKIIbeta, beta', and SSL exhibit robust and comparable interaction with CKIIalpha. Residues in SSL that mediate interaction with CKIIalpha appear similar to those in CKIIbeta, and SSL forms homodimers and heterodimers with CKIIbeta or beta' as well. We have tested all known Drosophila CKIIbeta-like proteins for rescue of the ion-homeostasis defect of yeast lacking beta subunits and find that CKIIbeta and SSL complement, beta' has marginal function, and Stellate appears non-functional. We have used real-time RT-PCR to assess developmental expression, and find that CKIIbeta is robust and ubiquitous, whereas SSL is restricted to males (third-instar-larvae, pupae, and adults), but is nondetectable in females of the corresponding stages. These results indicate that SSL expression encompasses a greater developmental window than that previously suggested and may confer distinct functions to CKII in a sex-specific manner. (C) 2003 Elsevier Science (USA). All rights reserved.