Nasopharyngeal cancer (NPC) constitutes a type of carcinoma encountered frequently in Southern China, among Eskimos of the Arctic region, and to a lesser extent in Southeast Asia. Because EBV DNA present in plasma or serum of NPC patients has proven to represent a promising noninvasive tumor marker, the present study was designed to determine the incidence of serum/plasma EBV DNA by nested PCR during various disease management stages. By this method, we could detect EBV DNA in plasma/serum of 98 of 167 NPC patients prior to treatment, compared with 10 of 77 samples derived from healthy blood donors serving as controls, with a similar prevalence observed in plasma versus serum. Investigation of 13 patients subjected to radiotherapy revealed plasma EBV DNA to persist in the plasma of one case, whereas among the remaining patients, it had vanished during the early phase of treatment. Finally, with 52 samples derived from 37 NPC patients during follow-up, we established 100% specificity and 0% false-positive rate for plasma DNA detection by nested PCR. Moreover, we subjected 24 known EBV DNA-positive serum samples to DNase digestion prior to DNA extraction and amplification to differentiate between free and encapsulated viral DNA, which demonstrated complete absence of the human beta-globin genomic DNA in contrast to EBV DNA detectable in 14 samples. In conclusion, applying this noninvasive method, serum/plasma EBV DNA constitutes a reliable tumor marker prior to, during, and after treatment of NPC.