The telomere and the enzyme telomerase in esophageal cancer have been poorly investigated. We present here aspects of the telomere and telomerase in esophageal cancer in relation to cell proliferation, differentiation and chemosensitivity to anticancer drugs. The telomere length (mean length of telomere restriction fragments; TRF), telomerase activity (TA), and human telomerase RNA (hTR) expression in a panel of 13 human esophageal cancer cell lines, squamous in origin, was examined by Southern blotting, the telomeric repeat amplification protocol (TRAP), and reverse transcriptase-polymerase chain reaction (RT-PCR), respectively. Cell proliferation expressed by the doubling time, cell differentiation determined by the keratin 13 and/or 14 expression, and chemosensitivity to cisplatin (CDDP) and 5-fluorouracil (5-FU) were compared with telomere-related factors. TRF shortening, the up-regulation of TA, and hTR expression was seen in all 13 cell lines. The TA correlated positively with the telomere length and negatively with the hTR expression. The doubling times of the cell lines and the telomere-related factors did not show any significant relation. The TA in the keratin 13/14-negative cell lines was significantly higher than that of the keratin 13-positive cell lines. The cells with short telomere tended to be resistant to CDDP whereas the cells with higher TA tended to be more sensitive to CDDP; 5-FU showed no relation to any telomere-related factors. Therefore, the activation of TA in esophageal squamous cell carcinoma is regulated by cell differentiation but not by cell proliferation, cells with high TA are more sensitive to CDDP, and cells with short telomere require a CDDP dose escalation.