We previously reported that LPS stimulation of the RAW264.7 murine macrophage cell line rapidly induced a structural change within the N terminus of the transcriptional regulatory factor PU.1. PU.1 is required for the expression of a variety of cytokine, cytokine receptor, and integrin genes. Western blot analysis of nuclear extracts prepared from LPS-stimulated macrophages revealed increased phosphorylation of PU.1 at serine residues relative to that in unstimulated controls. PU.1-DNA complexes prepared using nuclear extracts from LPS-stimulated macrophages were less sensitive to protease digestion compared with PU.1-DNA complexes generated using nuclear extracts prepared from unstimulated cells. This altered protease sensitivity probably reflects a conformational change within PU.1 resulting from LPS-induced phosphorylation. This possibility was supported by the finding that in vitro-phosphorylated PU.1 was similarly resistant to protease digestion. Transient transfection studies suggest that LPS-induced phosphorylation of PU.1 at serine 148, located within a casein kinase II (CKII) consensus motif, increases the transactivation function of PU.1. Other serine/CKII sites located at positions 41, 45, 132, and 133 do not appear to be required for LPS-induced PU.1 function. Lastly, we found that LPS also increased the enzymatic activity of CKII in these cells. To our knowledge, these are the first studies to demonstrate that LPS can stimulate CKII activity, induce PU.1 phosphorylation, and enhance the capacity of PU.1 to activate transcription in macrophages.