To study the specificity of nucleotide incorporation opposite 5-hydroxypyrimidines in template DNA, 18- and 45-member oligodeoxyribonucleotides, containing an internal 2'-deoxy-5-hydroxyuridine (5-OHdU) in two different sequence contexts, were used. Translesion synthesis past 2'-deoxy-5-hydroxyuridine (5-OHdU) in both oligonucleotides occurred, but pauses both opposite, and one nucleotide prior to, the modified base in the template is observed. The specificity of nucleotide incorporation opposite 2'-deoxy-5-hydroxyuridine (5-OHdU) in the template is sequence context dependent. In one sequence context, dA is the principal nucleotide incorporated opposite 2'-deoxy-5-hydroxyuridine (5-OHdU). However in a second sequence context, dC is the predominant base incorporated opposite 2'-deoxy-5-hydroxyuridine (5-OHdU). In that same sequence context, dC is also the predominant nucleotide incorporated opposite 2'-deoxy-5-hydroxyuridine (5-OHdU).