Phosphoprotein ppUL44 of the human cytomegalovirus (HCMV) DNA polymerase plays an essential role in viral replication, conferring processivity to the DNA polymerase catalytic subunit pUL54 by tethering it to the DNA. Here, for the first time, we examine in living cells the function of the highly flexible loop of ppUL44 (UL44-FL; residues 162 to 174 [PHTRVKRNVKKAP(174)]), which has been proposed to be directly involved in ppUL44's interaction with DNA. In particular, we use a variety of approaches in transfected cells to characterize in detail the behavior of ppUL44Deltaloop, a mutant derivative in which three of the five basic residues within UL44-FL are replaced by nonbasic amino acids. Our results indicate that ppUL44Deltaloop is functional in dimerization and binding to pUL54 but strongly impaired in binding nuclear structures within the nucleus, as shown by its inability to form nuclear speckles, reduced nuclear accumulation, and increased intranuclear mobility compared to wild-type ppUL44. Moreover, analysis of cellular fractions after detergent and DNase treatment indicates that ppUL44Deltaloop is strongly reduced in DNA-binding ability, in similar fashion to ppUL44-L86A/L87A, a point mutant derivative impaired in dimerization. Finally, ppUL44Deltaloop fails to transcomplement HCMV oriLyt-dependent DNA replication in cells and also inhibits replication in the presence of wild-type ppUL44, possibly via formation of heterodimers defective for double-stranded DNA binding. UL44-FL thus emerges for the first time as an important determinant for HCMV replication in cells, with potential implications for the development of novel antiviral approaches by targeting HCMV replication.