We have isolated a 27-kDa protein that binds to cytoplasmic dynein. Microsequencing of a 17-amino acid peptide of this polypeptide yielded a sequence which completely matched the predicted sequence of the beta subunit of casein kinase II, a highly conserved serine/threonine kinase. Affinity chromatography using a dynein column indicates that both the alpha and beta subunits of casein kinase II are retained by the column from rat brain cytosol. Although dynactin is also bound to the column, casein kinase II is not a dynactin subunit. Casein kinase II does not co-immunoprecipitate with dynactin, and it binds to a dynein intermediate chain column which has been preblocked with excess p150(Glued), a treatment that inhibits the binding of dynactin from cytosol. Bacterially expressed and purified rat dynein intermediate chain can be phosphorylated by casein kinase II in vitro. Further, native cytoplasmic dynein purified from rat brain can also be phosphorylated by casein kinase II in vitro. We propose that CKII may be involved in the regulation of dynein function possibly by altering its cargo specificity or its ability to interact with dynactin.