The loss of blood group antigen A on tumor tissue has been reported to be a strong adverse prognostic marker for patients with resected non-small cell lung cancer (NSCLC). Results have varied with respect to the prognostic significance of flow cytometric data. We sought to confirm the prognostic significance of blood group antigen A loss and flow cytometry in a large cohort of patients with early-stage NSCLC. Two hundred and sixty patients with surgically resected stage I (n = 193) and II (n = 67) NSCLC with at least a 5-year follow-up were identified. Using paraffin-embedded primary tumor, immunohistochemical stains for blood group antigen A were performed on 90 patients with blood type A or AB. The DNA index and percentage of cells in S phase were successfully obtained on 188 and 152 patients, respectively. The median survival time of the patients with primary tumors negative for blood group antigen A was 38 months (n = 36), compared with 98 months (n = 54) for those with antigen A-positive tumors (P < 0.01). The median disease-free survival times for antigen A-negative and -positive tumors were 26 months and 98 months, respectively (P < h 0.01). The median survival time of the patients with aneuploid tumors was 51 months (n = 131), compared with 50 months (n = 57) for those with diploid tumors (P = 0.42). The median survival time of the patients with S phase >8% was 44 months (n = 105), compared with 60 months (n = 47) for those with S phase </=8% (P = 0.18). Multivariate analysis showed that the loss of antigen A, higher N and T stages, and the presence of mucin predicted for poorer disease-free and overall survival. In the subgroup of patients with blood group A or AB, the loss of A antigen was the most powerful negative predictor of survival. Aneuploidy and percentage of cells in S phase were not of prognostic significance in this group of patients with resected stage I and II NSCLC. The value of blood group antigen A analysis needs to be evaluated in larger and prospective studies of early-stage NSCLC. Alteration of blood group antigen cell surface expression may represent an important marker for more aggressive biological and metastatic behavior in NSCLC.