DNA polymerase zeta (Pol zeta) and Rev1 are essential for the repair of DNA interstrand crosslink (ICL) damage. We have used yeast DNA polymerases eta, zeta and Rev1 to study translesion synthesis (TLS) past a nitrogen mustard-based interstrand crosslink (ICL) with an 8-atom linker between the crosslinked bases. The Rev1-Pol zeta complex was most efficient in complete bypass synthesis, by 2-3 fold, compared to Pol zeta alone or Pol eta. Rev1 protein, but not its catalytic activity, was required for efficient TLS. A dCMP residue was faithfully inserted across the ICL-G by Pol eta, Pol zeta, and Rev1-Pol zeta. Rev1-Pol zeta, and particularly Pol zeta alone showed a tendency to stall before the ICL, whereas Pol eta stalled just after insertion across the ICL. The stalling of Pol eta directly past the ICL is attributed to its autoinhibitory activity, caused by elongation of the short ICL-unhooked oligonucleotide (a six-mer in our study) by Pol zeta providing a barrier to further elongation of the correct primer. No stalling by Rev1-Pol zeta directly past the ICL was observed, suggesting that the proposed function of Pol zeta as an extender DNA polymerase is also required for ICL repair.