Clinical and experimental evidence has linked nutritional folic acid status to both anti- and procarcinogenic activity. Folate supplementation of normal cells appears to have a protective effect; however, folate supplementation of initiated cells may promote neoplastic progression. Given these considerations, the present series of experiments examines alterations in DNA metabolism and cumulative DNA lesions using an in vitro model of folate deprivation and repletion. DNA repair-deficient CHO-UV5 cells were cultured in Ham's F-12 medium or in custom-prepared Ham's F-12 medium lacking in folic acid, thymidine and hypoxanthine for a period of 18 days without cell passage. The results indicated that progressive folate and nucleotide depletion leads to a significant increase in the ratio of dUTP/dTTP and to the misincorporation of uracil into DNA. These alterations were accompanied by growth inhibition, DNA strand breaks, abasic sites and phenotypic abnormalities. After 14 days in culture, there was significant increase in gene amplification potential in the chronically folate-deficient cells, but no significant increase in anchorage-independent growth or in neoplastic transformation. Acute folate repletion of the deficient cells was used as a proliferative stimulus under conditions of dNTP pool imbalance and multiple lesions in DNA. A further increase in gene amplification was accompanied by anchorage-independent growth and neoplastic cell transformation as evidenced by aggressive tumor growth in Balb/c nu/nu mice. Using a sensitive in vitro model system, these results emphasize the essentiality of folic acid for de novo nucleotide synthesis and the integrity of the DNA. However, the in vivo relevance, especially in terms of tumorigenic potential, is not clear.