AIM: To investigate the expression of thymidylate synthase (TS) and glutathione-s-transferase π (GST-π) in esophageal squamous cell carcinoma and their association with the clinicopathologic characteristics. METHODS: Immunohistochemical methods were used to detect the expression of TS and GST-π in surgically resected formalin-fixed, paraffin-embedded esophageal squamous cell carcinoma (ESCC) tissue sections from 102 patients (median age, 58 years) and in 28 normal esophageal mucosa (NEM) samples. The relationship between TS and GST-π expression and clinicopathologic factors was examined. RESULTS: The expression of TS and GST-π was not statistically significantly associated with age of the patients, tumor size, lymph node metastasis, depth of invasion or tumor stage. TS staining was positive in 17.86% of normal esophageal mucosa and in 42.16% of ESCC samples (P ＜ 0.05). The expression level of TS was not only significantly lower in well-differentiated (21.88%) than in poorly-differentiated carcinomas (51.43%, P ＜ 0.05), but was also significantly higher in samples from male patients (46.51%) than from female patients (18.75%, P ＜ 0.05). GST-π was positively stained in 78.57% of normal esophageal mucosa and in 53.92% of ESCC samples (P ＜ 0.05). The expression level of GST-π was also significantly higher in welldifferentiated carcinomas (65.63%) than in poorlydifferentiated carcinomas (35.00%, P ＜ 0.05). CONCLUSION: The expression of TS and of GST-π may be used as molecular markers for the characterization of ESCC. Poorly-differentiated cells showed increased expression of TS and reduced expression of GST-π.