Human cells lacking DNA polymerase eta (pol eta) are sensitive to platinum-based cancer chemotherapeutic agents. Using DNA combing to directly investigate the role of pol eta in bypass of platinum-induced DNA lesions in vivo, we demonstrate that nascent DNA strands are up to 39% shorter in human cells lacking pol eta than in cells expressing pol eta. This provides the first direct evidence that pol eta modulates replication fork progression in vivo following cisplatin and carboplatin treatment. Severe replication inhibition in individual platinum-treated pol eta-deficient cells correlates with enhanced phosphorylation of the RPA2 subunit of replication protein A on serines 4 and 8, as determined using EdU labelling and immunofluorescence, consistent with formation of DNA strand breaks at arrested forks in the absence of pol eta. Pol eta-mediated bypass of platinum-induced DNA lesions may therefore represent one mechanism by which cancer cells can tolerate platinum-based chemotherapy.