We examined whether the shortening of telomere structure is related to in vitro cellular aging after multiple low-dose irradiation. We used three strains of HE cells (HE23, HE31, and HE40) exhibiting different levels of telomerase activity and irradiated these cells twice a week with a dose of 2 cGy or 4 cGy of X-rays until they senesced. The cells were in total exposed to doses of 52-208 cGy of X-rays. Only the HE31 cells, which had no telomerase activity, experienced an increase in the number of cell divisions, reaching a maximum of 120-124% of the non-irradiated controls. However, in two strains which did exhibit telomerase activity in an early passage in culture, no extension of cell life span was found. Telomerase-positive cells completely lost all telomerase activity when the cells were subcultured several times without irradiation. In the HE31 cells where the life span was extended, the ratio of cell having a long telomere was higher than those of the other two cells (HE23 and HE40). Cytogenetic analysis revealed that the life span extension due to multiple low-dose irradiation which was observed in HE31 cells did not correlate with specific chromosome alterations. Our results suggest that the telomerase activity remaining in the cells at an early passage does not correlate with the extension of life span in vitro by X-irradiation. The factor other than telomerase activity may play an important role in the regulation of telomere length and the extension of life span.