Recently we reported a differentiation-dependent inhibition of telomerase activity in human epidermis. Consistent with this observation we found that in keratinocyte cultures calcium-induced differentiation correlates with a decline in telomerase activity. To get further support for a role of calcium in the regulation of telomerase and to elucidate the underlying molecular mechanisms we investigated the effect of calcium on telomerase in the human epidermal keratinocyte line HaCaT. Treatment with thapsigargin, which increases intracellular calcium concentrations, inhibited telomerase activity without downregulating the expression of hTERT (human telomerase reverse transcriptase). This observation together with the fact that increasing calcium reduced telomerase activity in cell-free extracts, suggests that calcium directly interacts with the telomerase complex. This interaction could be mediated by the calcium-binding protein S100A8 as indicated by its ability to mimic the inhibitory effect of calcium. S100A8-induced reduction in telomerase activity was abrogated by S100A9. The ratio of both proteins remained constant in cells treated with thapsigargin, but their interactions were altered similarly in intact cells after thapsigargin treatment and in cell-free extracts in response to calcium. We hypothesize that calcium binds to S100A8/S100A9 complexes and alters their composition, thus enabling S100A8 to interact with the telomerase complex and inhibit its activity.