Protein kinase CKII (CKII) is a heterotetramer composed of two catalytic (alpha or alpha') and two regulatory (beta) subunits. Using the yeast two-hybrid system, we have identified the highly basic, ribosomal protein L41 as a cellular protein capable of interacting with the beta subunit of CKII. We show, furthermore, using purified proteins, that L41 protein and CKIIbeta associate directly in vitro. L41 protein is not a substrate for CKII phosphorylation, and it does not stimulate CKII activity with either beta-casein or synthetic peptide substrate (RRREEETEEE). However, L41 protein stimulates the phosphorylation of DNA topoisomerase IIalpha by CKII by 2.5 times. Additionally, L41 protein enhances the autophosphorylation of CKIIalpha. The data indicate that L41 protein associates with CKII and can modulate its activity toward a specific substrate or substrates. The direct interaction of CKIIbeta with ribosomal proteins also suggests that CKIIbeta itself or CKII holoenzyme may be involved in ribosome assembly or translational control.