Casein kinase II is thought to play an essential role in the control of cell division and differentiation in all eukaryotes. Through complementation of a defective casein kinase II catalytic subunit gene from Saccharomyces cerevisiae, we isolated an Arabidopsis thaliana casein kinase II regulatory subunit homologue, CKB1. A second regulatory subunit was identified by low-stringency hybridization with CKB1. Casein kinase II from S. cerevisiae is composed of two catalytic (alpha) and two regulatory (beta) subunits. Simultaneous disruption of the genes for the alpha and alpha' subunits, CKA1 and CKA2, respectively, is lethal. Strain YDH8 has disruptions of CKA1 and CKA2; its viability depends on a temperature-sensitive allele of CKA2, cka2-8, carried on a centromeric plasmid. We screened an A. thaliana cDNA library, whose inserts are under the control of the galactose-inducible GAL10 promoter, for cDNAs which enabled YDH8 cells to grow at the restrictive temperature. One cDNA, CKB1, was isolated by this screen which had homology to cDNAs of casein kinase II beta subunits. A second cDNA, CKB2, was isolated by hybridization and was also able to suppress the YDH8 mutant phenotype. The proteins encoded by CKB1 and CKB2 are 80% identical. The carboxy-terminal two thirds of both proteins is ca. 54% identical to the regulatory beta subunits of casein kinase II from other species. The amino termini are unrelated to any other known proteins. CKB1 and CKB2 lack the conserved autophosphorylation site characteristic of animal beta subunits, but have potential casein kinase II phosphorylation sites in the same region. Suppression of the cka1 delta cka2-8 mutant phenotype occurs by interaction of CKB1 with the defective, cka2-8-encoded, catalytic subunit. Cells with disruptions in CKA1 and CKA2 are not rescued by expression of CKB1.