Both HMECs and HMFs underwent a limited number of population doublings before reaching plateau. Morphologically, the cells enlarged, adopted a flattened shape with vacuolated cytoplasm and expressed the senescence-associated ?-galactosidase. Both cells types had low proliferative and apoptotic rates, as shown by BrDU incorporation and annexin V staining, respectively. In contrast to HMFs, which failed to produce proliferating cells from senescent cultures (even after 5 months), the senescent growth plateau in HMECs was transient. Small refractile proliferative cells emerged from this plateau to undergo a second period of exponential growth followed by a second plateau growth phase. Cells at the second plateau were quite different from those at the first, (senescent) plateau, showing greater heterogeneity and continuing to incorporate BrDU. A proportion (~20%) were also annexin V positive. They also lost expression of p16, a feature frequently associated with the development of epithelial malignancies. Analysis of metaphase spreads revealed many abnormalities at the cytogenetic level, including translocations, deletions, polyploidy, aneuploidy and telomeric associations. Telomere erosion, not seen in the senescent HMECs at the first plateau, was a feature of cells at the second plateau. This was also accompanied by a reduction in the number of telomeric repeats identified by FISH.