OBJECTIVE To identify novel genes that may participate in the pathogenesis of nasopharyngeal carcinoma (NPC). METHODS Sixty-three single copies of expressed sequence tag (EST) clusters mapped in the region of D3S1609-D3S1295 on chromosome 3p21-22 were obtained and assembled into long or full-length cDNA consensus sequence by in silico cloning strategy. Accordingly, these unigene consensus were studied by semi-quantitative reverse transcriptional (RT)-PCR with poorly differentiated squamous cell carcinoma tissues from 32 patients with primary nasopharyngeal carcinoma (NPC) and inflammatory tissues from 1b noncancerous individuals with chronic nasopharyngitis, as well as 4 NPC cell lines (CNE1bCNE2bHNE1 and HNE2). RESULTS Forty-nine unigene ESTs were expressed equally in both NPC and NP, 8 were expressed neither in NPC nor in NP, 2 had higher expression in NPC than in NP, and 4 had lower expression (or undetectable) in NPC than in NP. The expression of Hs.27566 gene was detected in none of the NPC cell lines, and was significantly reduced or undetectable in NPC biopsies in comparison with NP (P<0.001). CONCLUSIONS The differential expression of 63 novel genes located on chromosome 3p21-22 in NPC is mapped and 6 novel genes with differential expression in NPC tissues are identified.