1,2-dihydro-2-oxoadenine (2-OH-A), a common DNA lesion produced by reactive oxygen species, is a strong replicative block for several DNA polymerases (DNA pols). We have previously shown that various bases can be misincorporated opposite the 2-OH-A lesion and the type of mispairs varies with either the sequence context or the type of DNA pol tested. Here, we have analysed the ability of the human pol family X member DNA pol lambda, to bypass the 2-OH-A lesion. DNA pol lambda can perform error-free bypass of 2-OH-A when this lesion is located in a random sequence, whereas in a repeated sequence context, even though bypass was also largely error-free, misincorporation of dGMP could be observed. The fidelity of translesion synthesis of 2-OH-A in a repeated sequence by DNA pol lambda was enhanced by the auxiliary proteins Proliferating Cell Nuclear Antigen (PCNA) and Replication Protein A (RP-A). We also found that the DNA pol lambda active site residue tyrosine 505 determined the nucleotide selectivity opposite 2-OH-A. Our data show, for the first time, that the 2-OH-A lesion can be efficiently and faithfully bypassed by a human DNA pol lambda in combination with PCNA and RP-A.