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AIM: To investigate the effects of 8-Br-cAMP on differentiation and apoptosis of human esophageal cancer cell line Eca-109, and the related gene expression.METHODS: The cultured Eca-109 cells were divided into four groups: E1 group (co-cultured with 8-Br-cAMP for 24 h); E2 group (co-cultured with 8-Br-cAMP for 48 h); C1 group (treated without 8-Br-cAMP for 24 h); and C2 group (treated without 8-Br-cAMP for 48 h). The same concentration of cell suspension of each group was dropped separately onto the slides and nitrocellulose membranes (NCM). The biotin-labeled cDNA probes for c-myc, wild-type (wt) p53, bcl-2 and iNOS were prepared for in situ hybridization. The expressions of epidermal growth factor receptor (EGFR), p38 kinase, FAS, FasL and caspase-3 were detected using immunocytochemistry, and the NOS activity and the ratio of differentiated cells/proliferating cells were examined by cytochemistry. Immunocytochemistry, cytochemistry,and in situ hybridization were separately carried out on both slides and NCM specimens for each group. In addition, TUNEL was used to detect the cell apoptosis rate in each group.RESULTS: The apoptotic rate of E2 group was significantly higher compared to E1 group, while there was no difference in the ratio of differentiated cells/ proliferating cells between E1 and E2 groups. The signals of wt p53 and iNOS were markedly stronger, while the signals of c-myc and EGFR were obviously weaker in E1 group than those in C1 group (P＜0.05). Moreover, the signals of wt p53, iNOS, p38 kinase, caspase-3 and NOS activity were significantly stronger, whereas, the signals of bcl-2, c-myc and Fas/FasL were markedly weaker in E2 group than those in C2 group (P＜0.05). CONCLUSION: The differentiation and apoptosis of human esophageal cancer cell Eca-109 can be induced after 24- and 48-h treatment with 8-BrcAMP, respectively. Upregulation of wt p53, iNOS and downregulation of c-myc may be associated with differentiation and apoptosis of Eca-109 cells.Furthermore, upregulation of FasL, p38 kinase and caspase-3 as well as downregulation of bcl-2, and Fas may be involved in the apoptosis of Eca-109 cells.