Of the four subunits constituting DNA polymerase 6, subunit C or p66 has been shown to mainly mediate polymerase interaction with PCNA, an auxiliary factor that greatly enhances DNA polymerase 6 processivity on primed DNA templates. Here, we provide evidence that a highly conserved region located between amino acids 384 and 399 in the C-terminus of p66 is phosphorylated, most probably by Protein kinase CK2, and that another region, most probably located within the PCNA interacting domain in its extreme C-terminus, regulates its interaction with PCNA. Phosphorylation of p66 is associated with its co-localization with large subunit of DNA polymerase 6, p125, and PCNA, to the insoluble chromatin fraction at the beginning of S-phase. Taken together, the results provide evidence that concurrent phosphorylation events in p66 may positively and negatively regulate its activity and interactions with other components of the replisome during the cell cycle. (c) 2007 Elsevier Inc. All rights reserved.