The subunit composition of casein kinase II (CKII) from S. cerevisiae has been difficult to define, particularly with respect to the existence and number of regulatory (beta) subunits. A single, integral beta subunit, a loosely associated beta subunit, two distinct beta subunits, and a complete absence of beta subunits have all been proposed. Our laboratory reported yeast CKII to be composed of four polypeptides of 42, 41, 35, and 32 kDa (R. Padmanabha and C. V. C. Glover, 1987, J. Biol. Chem. 262, 1829-1835). The 42- and 35-kDa polypeptides were identified as distinct catalytic subunits, alpha and alpha', on the basis of N-terminal sequencing and subsequent molecular cloning. The 41- and 32-kDa polypeptides were found to undergo autophosphorylation, a characteristic of the beta subunit in other species, but antibodies raised against the beta subunit of Drosophila CKII crossreacted only with the 41-kDa polypeptide. In order to clarify the subunit composition of yeast CKII, particularly with regard to the 32-kDa polypeptide, we have purified the enzyme to homogeneity using a modified procedure. Based on the results of autophosphorylation studies, Western blotting, peptide mapping of the 41- and 32-kDa peptides, and sequencing of subunit-specific peptides, we demonstrate that the 32-kDa polypeptide is an additional beta subunit (beta') distinct from the 41-kDa beta subunit. This represents the first demonstration of beta subunit heterogeneity in purified CKII from any species.