Protein kinase CKII is a Ser/Thr kinase which is involved in many proliferation-related processes in the cell. P47(phox) is a component of the leukocyte NADPH oxidase, which is an important element of host defense against microbial infection. In this study, we demonstrate that a truncated form of the p47(phox) lacking its N-terminal region (p47(phox)/SH3-C), but not a truncated form of the P47(phox) lacking its C-terminal region (p47 (phox)/N-SH3), undergoes better phosphorylation by CKII in the presence of arachidonic acid. The yeast two-hybrid test and the glutathione S-transferase (GST) pull-down assay showed that p47(phox) interacts specifically with the regulatory beta subunit (CKII beta), but not with the catalytic a subunit (CKII alpha) of the tetrameric CKII holoenzyme. The binding of p47(phox) to CKII beta requires the C-terminal region of p47(phox) and is completely abolished by addition of spermine, indicating that a highly basic region in the C-terminal region of p47(phox) contributes to binding to CKII beta. In addition, p47(phox) stimulates the catalytic activity of CKII holoenzyme; this stimulation also requires the C-terminal region of p47. Coimmunoprecipitation experiments showed that CKII holoenzyme interacts with p47(phox) in human neutrophils. Taken together, the present data indicate that the C-terminal region of p47 ph,, plays a significant role in the arachidonic acid-dependent phosphorylation of p47(phox) by CKII and that the same region of p47(phox) associates directly with CKII beta and can modulate the catalytic activity of CKII holoenzyme.