Several studies have described p16INK4A and prostaglandin E2 (PGE2) co-alterations in various solid tumors, including non-small cell lung cancer (NSCLC). In this study, we examined the correlation between PGE2 receptor 2 (EP2) expression and p16INK4A methylation in NSCLC, and the association with clinicopathological features and prognostic significance. We retrospectively reviewed 88 NSCLC patients who underwent resection from July 1993 to May 1997. The tumors included 43 adenocarcinomas, 39 squamous cell carcinomas, and 6 large cell carcinomas. EP2 expression was determined by immunostaining, and p16INK4A methylation was analyzed by methylation specific PCR. EP2 was overexpressed in 44% of NSCLC patients, 61% of adenocarcinoma cases, 28% of squamous cell carcinoma cases, and 33% of large cell carcinoma cases. EP2 expression positively correlated with lymph node metastasis (P=0.034), especially in patients with squamous cell carcinoma (P<0.009). Methylation of p16INK4A was detected in 34% of NSCLC patients, 23% of adenocarcinoma cases, 44% of squamous cell carcinoma cases, and 50% of large cell carcinoma cases. In patients with squamous cell carcinoma, EP2 overexpression correlated with poor prognosis with a relative risk of 2.4 (confidence interval 2.1-51.8, P<0.003), and positively correlated with p16INK4A methylation (P<0.024). Adenocarcinoma patients with p16INK4A methylation had poor prognosis with a relative risk of 2.4 (confidence interval 1.8-69.7, P<0.009), but this was not correlated with EP2 expression. In conclusion, EP2 overexpression was common in NSCLCs, especially in adenocarcinomas. Synchronous alteration of p16INK4A and EP2 may accelerate progression of squamous cell carcinomas. These two alterations may differentially affect pathogenesis among subtypes of NSCLC.