Wild-type p53 is necessary for the growth arrest of human diploid fibroblasts (HDF) (and many other cell types) at the end of their proliferative lifespan. Although p53 may actively mediate senescence, possibly in response to telomere erosion, it is however equally possible that it is merely a permissive factor required for response to some other inducer. To address this question, we have generated stable transfectants of early passage HDF, represented here by clone LacZ21, in which expression of a beta-galactosidase reporter construct reflects p53 transactivation activity. During continuous passage, the proportion of beta-gal positive LacZ21 cells remained below 2% for 25 population doublings (pd), first became significantly increased after 29 pd, and thereafter increased rapidly, reaching a maximum of 88% in fully-senescent cells (32 pd), which exceeded the response observed following an optimum dose (20 J/m2) of u.v. radiation. Correspondingly, the proportion of cells incorporating bromodeoxyuridine (BrdU) (initially 45-50%) began to fall at 29 pd and thereafter dropped rapidly to below 1% by pd 32. There was therefore a near-perfect reciprocal relationship between reporter construct expression and DNA synthesis as cells approached senescence. Furthermore, a dominant-negative p53 mutant (introduced by retroviral transduction) rescued LacZ21 cells from senescence and generated colonies with extended lifespan in which beta-gal expression was totally abolished. These data, although not excluding the need for other p53 functions, strongly suggest that p53-mediated transactivation of growth regulatory genes is a direct trigger, rather than a permissive factor, for cellular senescence.