Ochratoxin A (OTA), a widespread food contaminant and potent renal carcinogen in rodents, is weakly genotoxic in mammalian cells. The mechanisms underlying OTA-induced genetic damage are still poorly understood. In its recent risk assessment, the European food safety authority (EFSA) considered that the specific spectrum of mutations and chromosomal damage induced by OTA may derive from unresolved replication stress. The aim of the present work was to experimentally test the hypothesis that OTA interferes with DNA replication and to characterize the cellular response to OTA-mediated replication stress. Using the DNA fiber assay to study replication fork dynamics at single molecule resolution, a small but statistically significant global delay in replication fork progression was observed in human kidney (HK-2) cells exposed to OTA at ≥ 10 µM. OTA-mediated interference with DNA replication was confirmed by a concentration-related decrease in incorporation of the thymidine analog 5-ethynyl-2'-deoxyuridine (EdU) into newly replicating DNA in HK-2 cells arrested in late G