In this work, we address the question of whether replicative senescence can be induced in immortal nontumorigenic human fibroblasts. The immortal fibroblasts used in this study were derived from two Li-Fraumeni (LF) patients who carry in their germ line one wild-type and one mutant p53 allele. Both immortal lines have lost the wtp53 allele and express no detectable p16INK4a protein, although they carry the p16INK4a gene. In contrast to immortal human fibroblasts, senescent human fibroblasts have a low content of 5-methyl-cytosine in their DNA. This observation suggested the possibility that a demethylating agent could revert the immortal phenotype and induce replicative senescence in the immortal cell lines. Cells of the two LF lines were exposed to the demethylating agent 5-aza-2'-deoxycytidine. Within 6 days, all cells were growth arrested and showed the enlarged and flat morphology characteristic of senescent cells, an accumulation of lipofuscin granules and senescence-associated beta-galactosidase activity at pH6, both biomarkers for senescence. Immunoblots of 5-aza-2'-deoxycytidine-treated cells showed a greatly increased expression of p16INK4a protein but no detectable change in the expression of p21CIP1, a gene known to be strongly expressed in senescent normal human fibroblasts. In two other experimental series, cells of the two LF lines were infected with retroviral constructs encoding either p16INK4a or p21CIP1. Each of the transduced genes induced senescence without affecting the expression of the other endogenous gene. The results show that induction of senescence in immortal LF fibroblasts can occur by different pathways: (a) by demethylation-dependent pathways that induce the expression of p16INK4a; and (b) by demethylation-independent pathways involving the expression of p21CIP1. The induction of senescence by p16INK4a and p21CIP1 occurred equally in the two human immortal fibroblast lines, which differed in the length of their telomeres and the activity of their telomerase.