Abstract Purpose: Folic acid and homocysteine levels are influenced by the circulating levels of estradiol. Little is known about the behavior of these metabolites during an in vitro fertilization cycle, where superovulation protocols induce a major increased in the levels of estradiol and other hormones. Methods: We performed a prospective, comparative, transversal, descriptive and observational study that includes 49 cases (group A), which were patients that entered a fertility program, and 14 controls (group B), which correspond to the donor group. In both groups, the levels of folic acid, estradiol and homocysteine as well as the MTHFR polymorphism were determined. Results: None of the patients has subnormal levels of folic acid or homocysteine and these levels were not modified after ovarian stimulation. We observed that homocysteine concentration tended to decrease with elevated estradiol levels produced by the stimulation of the ovary. The presence of allele C677T MTHFR was high in the populations evaluated in this study, with a particular high incidence of the TT allele. Conclusion: The levels of plasma and intracellular folate, and homocysteine do not change during hormonal stimulation.