Inflammation in the nasopharynx is very common worldwide and nasopharyngeal carcinoma (NPC) results in significant morbidity and mortality in southeast Asia and north Africa. To facilitate the understanding of pathogenesis of these diseases as well as normal nasopharynx biology, transcriptional gene expression profile of normal human nasopharynx, from undissected biopsies, was established in this study by generating a large amount of ESTs, followed by bioinformatics analysis. A total of 27,209 ESTs generated from human nasopharynx cDNA library were integrated into 8,420 non-redundant clusters, of which, 6,070 (72.09%) corresponded to known genes, 156 (1.85%) to known ESTs, and 2,194 (26.06%) to novel sequences, respectively. Functional classification revealed that transcripts constituting enzymes (2,284, 28.30%) and those participating in cell growth/maintenance (3,306, 46.33%) were the largest population expressed in the nasopharynx, followed by genes coding for binding proteins (2,135, 26.45%) and involved in cell communication process (1,832, 25.68%). In addition, through comparison of the nasopharynx gene expression profile with those of 7 other human tissues, 59 transcripts preferentially expressed and 22 transcripts unique in the nasopharynx were identified. Our study acquired an initial assessment of qualitative diversity of gene expression in the nasopharynx, providing the first step toward comprehensive characterization of the molecular features of the nasopharyngeal disorders.