Ectopic expression of telomerase blocks both telomeric attrition and senescence, suggesting that telomeric attrition is a mitotic counting mechanism that culminates in replicative senescence. By holding human fibroblast cultures confluent for up to 12 weeks at a time, we confirmed previous observations and showed that telomeric attrition requires cell division and also, that senescence occurs at a constant average telomere length, not at a constant time point. However, on resuming cell division, these long-term confluent (LTC) cultures completed 15-25 fewer mean population doublings (MPDs) than the controls prior to senescence. These lost divisions were mainly accounted for by slow cell turnover of the LTC cultures and by permanent cell cycle exit of 94% of the LTC cells, which resulted in many cell divisions being unmeasured by the MPD method. In the LTC cultures, p27(KIP1) accumulated and pRb became under-phosphorylated and under-expressed. Also, coincident with permanent cell cycle exit and before 1 MPD was completed, the LTC cultures upregulated the cell cycle inhibitors p21(WAF) and p16(INK4A) but not p14(ARF) and developed other markers of senescence. We then tested the relationship between cell cycle re-entry and the cell cycle-inhibitory proteins following subculture of the LTC cultures. In these cultures, the downregulation of p27(KIP1) and the phosphorylation of pRb preceded the complete resumption of normal proliferation rate, which was accompanied by the down-regulation of p16(INK4A). Our results show that most normal human fibroblasts can accumulate p16(INK4A), p21(WAF) and p27(KIP1) and senesce by cell division-independent mechanism(s). Furthermore, this form of senescence likely requires p16(INK4A) and perhaps p27(KIP1).