The presence of telomerase has been demonstrated recently in many different malignancies. Several reports documented that in human hepatocellular carcinoma, the level of telomerase activity parallels its differentiation stage. In the present study, the effect of the differentiation-inducing agent sodium butyrate on telomerase activity in four human liver cancer cell lines was investigated using the telomeric repeat amplification protocol. We assayed telomerase activity before and after butyrate treatment and in cell cycle synchronized non-dividing quiescent cells. In addition, telomerase reverse transcriptase levels were measured at the mRNA level. All four cell lines possessed high but not identical levels of telomerase activity. Telomerase activity was significantly reduced by treatment with sodium butyrate as well as trichostatin A in a dose- and time-dependent fashion, paralleling the reduction of cell proliferation. Although methotrexate, hydroxyurea, and colchicine synchronized the cell cycle at G1, S, and G2/M, respectively, and thereby also caused proliferating cells to cease dividing and become quiescent, in this case telomerase activity remained essentially unaltered compared to the control cultures. Moreover, levels of mRNA encoding telomerase reverse transcriptase were not always significantly altered by either sodium butyrate treatment or cell cycle synchronization. These results suggest that sodium butyrate, as a histone deacetylase inhibitor, effectively reduces telomerase activity without affecting transcription levels of the reverse transcriptase component. © 2001 Wiley-Liss, Inc.