Background: Nasopharyngeal carcinoma (NPC) is a prevalent cancer in Indonesia, usually identified at late stage. Epstein-Barr virus (EBV) gene products are present in all tumor cells. Early-stage NPC diagnosis is clinically important but difficult due to non- specific symptoms. In patients suspected of having NPC, nasopharyngeal (NP) brush-cytology may provide a simple non-invasive procedure for rapid on-site detection of NPC tumour cells that can be combined with EBV markers. Methods: Patients (N=83) suspected of having NPC underwent nasoendoscopy-guided NP brushing before a biopsy from clinically suspicious areas. The loaded brush was first smeared onto glass slides that were directly immersed in ethanol, followed by Papanicolaou (PAP) staining and on-site interpretation of tumour cell presence. Thereafter, slides were used for EBER RNA in situ hybridization (EBER-ISH) and LMP-1 immunocytochemistry (ICC). The brush left-over was used for EBV-DNA load measurement using real-time PCR. Biopsy specimens were taken from the same NP site and processed for routine pathology and EBER-ISH on tissue sections. Result: Morphological detection of NPC tumour cells on PAP-stained brush-smears gave a sensitivity and specificity of 87% and 100%, respectively. EBER-RISH and LMP1 ICC combined showed 100% positivity for NPC cases. Expression of LMP-1 was detected in 77.8% of cases. Median EBV-DNA load in left-over brush material from NPC cases was 5.06 × 106 copies/brush (range 777 to 242,400,000 copies/brush), whereas median value in non-NPC cases was 4.4 x 103 copies/brush (range 484 - 297,200; P < 0.001). Conclusion: Minimal invasive NP-brush cytology with conventional PAP staining allows rapid, simple and cheap “on site” detection of NPC tumour cells in suspected NP lesions. The method can be modified for direct demonstration of EBV involvement using EBER-ISH and LMP1-ICC or EBV-DNA load measurement. This NP brush procedure may be useful for primary screening in patients considered at risk for NPC.