There are metabolic and epidemiologic data consistent with the hypothesis that folate deficiency increases the likelihood of cancer. Conversely, it is also known that folate is necessary for cancer growth, but few experiments in laboratory animals have evaluated the effects of folate deficiency on the development of chemically induced cancers. Our purpose was to determine the effects of nutritional folate deficiency in female Fischer 344 rats on initiation and early promotion of methylnitrosourea (MNU)-induced mammary cancer. Rats (age, 27 days) were fed a folic acid-deficient diet (AIN-76A) supplemented with glycine and succinylsulfathiazole [FA(0)]; the FA(0) diet supplemented with 2 or 40 mg of folic acid per kilogram [FA(2) or FA(40), respectively]; or the FA(0) diet supplemented with 20 mg of folinic acid per kilogram [FL(20)]. At 57 days of age, each diet-treated group (30 rats in each group) received MNU (50 mg/kg) by intravenous injection. Immediately after MNU treatment, all animals were fed the AIN-76A complete diet containing 2 mg of folic acid per kilogram. Control groups were fed the AIN-76A complete diet throughout the entire experiment. After 4 weeks, folate deficiency, but not anemia or growth suppression, was documented by lower folate levels in plasma and red blood cells in the group receiving the FA(0) diet. Cancer multiplicity (i.e., number of mammary cancers per number of tumor-bearing animals) at 180 days after MNU injection was 1.32, 1.90, 2.14, and 2.73 mammary cancers per tumor-bearing animals in the FA(0), FA(2), FA(40), and FL(20) groups, respectively; the value in the FA(0) group was statistically significant compared with the values in the other groups. The time required for 50% of the rats to develop palpable mammary cancer was 170, 142, 100, and 85 days, respectively. The value of 170 days for the FA(0) group was statistically significant compared with the values of 100 and 85 days. Mammary cancer incidence was 63%, 70%, 72%,and 73%, respectively; these percentages were not significantly different. Folate deficiency suppresses and folate supplementation enhances initiation or early promotion of MNU induced mammary cancer in rats, even when the folate-deficient rats do not have anemia or growth suppression. Implication: Since the rat is relatively resistant to folate deficiency anemia, other animal models should be used to test the effect of folate nutriture on carcinogenesis. [J Natl Cancer Inst 84: 1740–1744, 1992]