The relationship of thyroid transcription factor-1 (TTF-1) and HER2/neu expression in non-small cell lung cancer (NSCLC) with multiple parameters including survival were examined. Patients with primary NSCLC who had surgical resection and follow-up of at least 5 years were included in the study. There were 57 patients (38 men and 19 women), 44 to 75 years old (median age, 61 years); 28 patients had adenocarcinoma (AD) and 29 had squamous cell carcinoma. Tumors were examined for TTF-1 and HER2/neu expression using formalin-fixed, paraffin-embedded tissue. Clinical and follow-up data were obtained from the hospital records and Cancer Center database. Representative tumor sections were stained using standard immunohistochemical technique and commercial antibodies for TTF-1 (clone 8G7G3/1, Dako) and HER2/neu (polyclonal, Dako). Tumors were graded as negative (<5%), weak positive (5-49%), and strong positive (>50%), based on the percentage of positively stained tumor cells. Statistical analyses were performed using log-rank test, Pearson and Spearman correlations, and Kaplan-Meier survival curves. TTF-1 expression was seen in 45.6% of all tumors (80% of ADs and 14% of squamous cell carcinomas). Eighteen patients with tumors showing strong TTF-1 expression had significantly better survival compared with the 39 patients whose tumors showed negative or weak TTF-1 expression, although many more of the higher stage AD had strong TTF-1 staining than stage I AD. The TTF-1 expression did not correlate with tumor differentiation and was considered an independent predictor of survival. Seventeen of the 18 tumors with strong TTF-1 expression were ADs. Only eight of 57 (17%) tumors showed HER2/neu expression; seven of these eight were ADs. Although HER2/neu expression and survival did not show correlation, the majority of those ADs with weak or strong HER2/neu staining also had strong TTF-1 staining, were mostly stage I tumors. and had overall longer survival. All patients with stage I disease showed better 5-year survival compared with those with stages II and III. Hispanic patients had significantly worse survival compared with Caucasians and African Americans. The results of this study suggest that strong expression of TTF-1 is an independent predictor of better survival and may be a useful prognostic tool for evaluation of patients with NSCLC.