The minor base 5-methylcytosine (5mC) in DNA may be important for the regulation of gene expression. Random loss of 5mC may occur during pre-replicative DNA synthesis in mortal cell strains, and thus give rise to biochemical aberrations in aging cells. 5-Azacytidine (5azaC) was used to induce loss of 5mC in DNA of human diploid fibroblasts (MRC-5) in an attempt to accelerate in vitro senescence. The 5mC content of DNA was measured by incorporation of [³H]uridine into dividing cells, hydrolysis of DNA and separation of bases by HPLC. In untreated MRC-5 cells, 5mC was 3.6% of the total cytosine (C+5mC) at population doubling (PD) 20 (28% of lifespan) and fell to 1.6% at PD 67 (97% of lifespan). A single pulse treatment with 5azaC (1 μg/ml) induced demethylation and shortened the lifespan by 10% (6.8 PDs loss). Pulse-treated cells showed temporary growth inhibition, though they subsequently regained normal growth rate and morphology. However, uniform treatment with 0.1 μg/ml 5azaC between PD 20 and 23 produced no immediate growth inhibition, but a 22% loss of 5mC and 25% decrement in lifespan (16.6 PDs loss). The present results indicate that 5mC levels fall during normal aging of MRC-5 cells and accelerated 5mC loss shortens the in vitro lifespan of these cells. Hypomethylation may thus be responsible for some aspects of in vitro aging.