Several strategies have been described for the primary culture of human myometrial cells. However, primary cultures of myometrial cells have a limited life span, making continual tissue acquisition and cell isolation necessary. Recent studies have demonstrated that cell culture life span is related to chromosomal telomere length, and cellular senescence results from progressive telomere shortening and the lack of telomerase expression. Transfection of cells with expression vectors containing the human telomerase reverse transcriptase (hTERT) maintains telomere length and effectively gives normal cells an unlimited life span in culture. In addition, hTERT extends the life span of cultured cells far beyond normal senescence without causing neoplastic transformation. In the present study, we developed a cell line from hTERT-infected myometrial cells (hTERT-HM). Cells were isolated from myometrial tissue obtained from women undergoing hysterectomy, and retroviral infection was used to express the catalytic subunit of telomerase in myometrial cells. Cells expressing hTERT have been in continuous culture for >10 mo, whereas the control culture senesced after approximately 2 mo. Telomerase activity was monitored in cells with a polymerase chain reaction-based telomerase activity assay. Telomerase-expressing cells contained mRNA for α smooth muscle actin, smoothelin, oxytocin receptor, and estrogen receptor α, but the estrogen receptor β receptor was lost. Immunoblotting analysis identified the expression of calponin, caldesmon, α smooth muscle actin, and oxytocin receptor. Although estrogen receptor expression was below the level of detection with immunoblotting, transfection experiments performed with reporter constructs driven by estrogen response elements demonstrated estrogen responsiveness in the hTERT-HM. In addition, treatment of hTERT-HM with oxytocin caused a concentration-dependent increase in intracellular calcium levels, confirming the presence of functional oxytocin receptors. Myometrial cells immortalized with hTERT retained markers of differentiation that are observed in primary cultures of smooth muscle cells. The expression of various smooth muscle/myometrium cell markers suggests that these cells may be an appropriate model system to study certain aspects of human myometrial function.