Casein kinase II (CKII) usually exists as a heterotetramer with alpha 2 beta 2, alpha alpha'beta 2, or alpha'2 beta 2. The alpha or alpha' subunits catalyze protein phosphorylation, whereas the function of the beta subunit remains unclear. One of the possible functions of the beta subunit may be to mediate the interaction of the catalytic subunit with target proteins. To identify proteins capable of associating with the beta subunit in vivo, we have used a two-hybrid system. One protein identified is human ribosomal protein L5. The protein L5 does not interact with the alpha or alpha' subunits of CKII, supporting the idea that the beta subunit can determine a substrate specificity of CKII. These results furthermore suggest a novel role for CKII in ribosomal L5 phosphorylation, in ribosomal assembly, or ribosomal transport in the intact cells. The protein L5 may act as a regulator of the activity or subcellular localization of CKII.