The Rad6-Rad18 ubiquitin-conjugating enzyme complex promotes replication through DNA lesions by means of at least three different pathways: the DNA polymerase (Pol) eta- and zeta-dependent translesion DNA synthesis (TLS) and a Rad5-Mms2-Ubc13-dependent pathway. In DNA-damaged yeast cells proliferating cell nuclear antigen (PCNA) becomes monoubiquitylated at the K164 residue, and genetic studies in yeast have indicated a requirement for this modification in TLS mediated by Poleta and Polzeta. To be able to decipher the role of PCNA monoubiquitylation in the TLS process, we have reconstituted this PCNA modification in vitro from purified yeast proteins. We show that, in addition to the requirement for Rad6-Rad18, the reaction depends on the loading of the PCNA homotrimeric ring onto the DNA by replication factor C and that all three PCNA monomers become efficiently ubiquitylated. The availability of PCNA monoubiquitylated on all of its three monomers has enabled us to examine the effects of this PCNA modification on DNA synthesis by Pols delta, eta, zeta, and Rev1. Contrary to the prevailing ideas that presume a role for PCNA ubiquitylation in the disruption of Poldelta's binding to PCNA or in the enhancement of the binding affinity of the TLS Pols for PCNA, we find that PCNA ubiquitylation does not affect any of these processes. These observations lead us to suggest a role for PCNA monoubiquitylation in disrupting the PCNA binding of a protein(s) that otherwise is inhibitory to the binding of PCNA by TLS Pols.