Both increased copper and reduced folate levels are commonly found in patients with liver diseases. To better understand the mechanisms by which folate deprivation interacts with copper to contribute to hepatocellular toxicity, rat primary hepatocytes were isolated, cultured in folate-deprived (FD) RPMI medium, and assayed for cytotoxicity after copper sulfate (CuSO4) exposure. MTT measurement and trypan blue assay showed that elevated CuSO4 levels aggravated cell death of folate-deprived but not folate-sufficient hepatocytes. CuSO4 treatment increased the levels of intracellular reactive oxygen species (ROS) by 3 times in FD hepatocytes and tripled the proportion of FD hepatocytes with hypodiploid DNA contents. Measurement of membrane phosphatidylserine exposure indicated that the CuSO4-mediated toxicity in FD hepatocytes was not mediated by the apoptotic pathway. Real-time polymerase chain reaction (PCR) analysis revealed that CuSO4 treatment did not increase the occurrence of a 4834-bp mtDNA (mtDNA4834) deletion in FD hepatocytes. Preincubation of FD hepatocytes with various concentrations of folate prior to CuSO4 treatment did not modulate the mtDNA4834 deletion. Taken together, the data suggest that elevated copper levels potentiate cell death of folate-deprived hepatocytes, which is primarily associated with increased ROS generation and chromosomal DNA loss. The cytotoxicity exerted by folate depletion and elevated copper levels, however, is not due to apoptosis or accumulated mtDNA4834 deletions in primary hepatocytes.