Casein kinase II (CKII) is thought to regulate a broad range of transcription factors, but its mode of action is not well characterized. We previously showed that CKII is co-purified with the ATF family of transcription factors using DNA-affinity latex beads. Here we report a functional and physical interaction between CKII and transcription factors. We demonstrate that CKII binds through its catalytic alpha and alpha' subunits to the basic leucine zipper (bZIP) DNA-binding domains of many transcription factors, including ATF1. Kinetic analysis using a surface plasmon resonance sensor suggests that CKII loosely associates with ATF1 in vivo . Deletion of the bZIP domain of ATF1 markedly reduces its phosphorylation by CKII, suggesting that the bZIP recruits CKII to the vicinity of the target site. ATF1-CKII complex is also formed on DNA. Using CKIIalpha fusedto a heterologous DNA-binding domain, we also demonstrate that CKII, when bound to DNA, efficiently phosphorylates its substrate, which is bound to the same DNA molecule. Taken together, CKII may regulate transcription (and possibly other events) by phosphorylating proteins on DNA.