Telomerase activity was measured in isolated cells from bovine and human adrenal cortex, in cells in primary culture, in cells in later passages in culture, and in cells genetically modified by expression of hTERT (human telomerase reverse transcriptase). Telomerase activity in freshly isolated bovine adrenocortical cells and in human adrenal cells from donors of various ages (6-79 years) was very low or undetected. However, primary bovine adrenocortical cell cultures were strongly positive for telomerase activity, and primary human adrenocortical cell cultures were weakly positive. Both cell types proliferate in primary culture but proliferation of bovine cells is much more vigorous. When primary bovine cells were subcultured to make successively secondary and tertiary cultures, telomerase activity declined strongly, and was undetected by the third passage. There was only a slight decrease in growth rate over this period. Levels of the telomerase RNA component did not change with passage number when assessed by semi-quantitative competitive RT-PCR. When both bovine and human cells were infected with a retrovirus encoding hTERT, telomerase activity in the cells was very high. We conclude that in the adrenal cortex, as in some other tissues, TERT expression is regulated and upregulation of telomerase activity is associated with rapid proliferation in primary culture. Telomerase activity is not maintained, and introduction of TERT is required for stable telomerase activity and for immortalization.