Nasopharyngeal carcinoma (NPC) is closely related with Epstein-Barr virus (EBV). Almost every NPC tumor cells carries clonal EBV genomes. Detection of EBV derived latent membrane protein-1 gene (LMP-1) indicate the presence of NPC. Middle ear effusion (MEE) is a frequent sign of NPC. There have been no reports on LMP-1 in MEE. Tympanocentesis of 88 ears with MEE of 66 patients were done in three groups of patients, group (I) NPC, 31 patients, 50 ears, (II) other head and neck cancers, five patients, six ears and (III) no cancer history, 30 patients, 32 ears. The middle ear aspirate and nasopharyngeal swab specimen were collected to detect LMP-1 with a PCR-based method. Sixty aspirates (68%) out of 88 ears with MEE had enough DNA for PCR amplification. LMP-1 was detected in six middle ear aspirate specimen from three patients in group I who had petrous apex invasion. LMP-1 was detected in 30 swab specimen (93.8%) out of 32 nasopharyngeal swabs in group I. LMP-1 was not detected in middle ear aspirates or nasopharyngeal swab in group II and III patients. LMP-1 was not detected in MEE in patients without NPC. In NPC patients, the detection of LMP-1 may indicate petrous apex invasion.