Activation of the ERK1/2 pathway is involved in malignant transformation both in vitro and in vivo. Little is known about the role of activated ERK1/2 in non-small cell lung cancer (NSCLC). The purpose of this study was to characterise the extent of the activation of ERK1/2 by immunohistochemistry in patients with NSCLC, and to determine the relationship of ERK1/2 activation with clinicopathological variables. Specimens from 111 patients with NSCLC (stages I-IV) were stained for P-ERK. Staining for epidermal growth factor receptor (EGFR) and Ki-67 was also performed. In all, 34% of the tumour specimens showed activation for ERK1/2, while normal lung epithelial tissue was consistently negative. There was a strong statistical correlation between nuclear and cytoplasmic P-ERK staining and advanced stages (P<0.05 and P<0.001, respectively), metastatic hilar or mediastinal lymph nodes (P<0.01, P<0.001), and higher T stages (P<0.01, P<0.001). We did not find correlation of nuclear or cytoplasmic P-ERK staining with either EGFR expression or Ki-67 expression. Total ERK1/2 expression was evaluated with a specific ERK1/2 antibody and showed that P-ERK staining was not due to ERK overexpression but rather to hyperactivation of ERK1/2. Patients with a positive P-ERK cytoplasmic staining had a significant lower survival (P<0.05). However, multivariate analysis did not show significant survival difference. Our study indicates that nuclear and cytoplasmic ERK1/2 activation positively correlates with stage, T and lymph node metastases, and thus, is associated with advanced and aggressive NSCLC tumours.