Purpose and Experimental Design: The molecular mechanisms by which the pl4ARF gene is altered in non-small cell tang cancer (NSCLC) are complex and unclear. Using genetic and epigenetic analyses, we examined various molecular alterations including the loss of protein and mRNA expression, and 5′CpG hypermethylation, allelic imbalance, and mutation of the p14ARF gene in a series of 102 NSCLC samples, in parallel with clinicopathological and prognostic analyses. To clarify the biological significance of p14ARF alterations, its relationship with p16INK4a and p53 alterations was also examined. Results: We found that 34% of NSCLC patients had aberrant P14ARF protein expression, which was more frequent in adenocarcinomas (AD; 44%) than in squamous cell carcinomas (22%; P = 0.024). A high concordance was observed between alterations in protein and mRNA expression and 5′CpG hypermethylation (P ≤ 0.001). The pl4ARF hypermethylation inversely correlated with P53 overexpression (P = 0.001). This mutually exclusive relationship for alteration between p14ARF and p53 was also supported by a worse prognosis of AD patients with positive P14ARF expression (P = 0.01) and of AD patients with P53 overexpression (P = 0.006). Our data also indicated that hemizygous/ homozygous deletion and mutation in the p14ARF gene occurred at 26%, 9%, and 0%, respectively, of microdissected NSCLCs. Conclusions: Our data suggest that p14ARF 5′CpG hypermethylation is the predominant mechanism involved In the aberrant expression of the pl4ARF gene. In addition, p14ARF 5′CpG hypermethylation occurs inversely to P53 overexpression.