We investigated thymidylate synthase (TS) expression in tumor tissues and examined the relationship between TS expression and post-operative survival in patients with p-stage I adenocarcinoma of the lung. A total of 104 patients, who underwent complete resection for p-stage I adenocarcinoma of the lung, were retrospectively reviewed. TS expression in tumor tissues was evaluated by immunohistochemical staining using rhTS polyclonal antibody. The intensity of immunohistochemical staining was classified into four categories using a visual grading system from 0 to 3. The percentage of each grade of TS staining was 9.6% for Grade 0, 18.3% for Grade 1, 35.6% for Grade 2 and 36.5% for Grade 3. Five-year survival rates of patients with Grade 0 to Grade 3 were 90.0, 83.9, 70.3 and 73.7%, respectively with no significant difference among all groups (P=0.236). When divided into two groups, according to the intensity of the grade, 5-year survival rates of TS low expression group (Grade 0 and Grade 1) and TS high expression group (Grade 2 and Grade 3) were 86.1 and 72.0%, respectively, with a significant difference (P=0.048). In conclusion, high level of TS expression was associated with poor prognosis. Immunohistochemical evaluation of TS expression may be useful to predict survival after complete resection in p-stage I adenocarcinoma of the lung.