The levels of intracellular ribonucleotide reductase activity, a highly regulated rate-limiting step in DNA synthesis, were investigated during serial subculture of normal human diploid fibroblasts in vitro. This key enzyme activity was found to decline significantly during cellular senescence. This observation along with previous findings of a mutator gene associated with mammalian ribonucleotide reductase suggests a possible mutation mechanism for aging which involves changes in reductase activity during cellular senescence. Furthermore, in keeping with the decrease in enzyme activity, we show that cell resistance to the antitumor agent hydroxyurea, whose site of action is ribonucleotide reductase, decreases progressively with increasing passage numbers. This indicates that an important factor to be considered in drug therapy aimed at the reductase is the increased sensitivity of normal cells to drug with cell age, due to a decline in enzyme activity. Much remains to be determined about age-dependent factors involved in drug therapy; cultured normal human diploid fibroblasts provide a useful system in which to investigate these important parameters.