Objective: To investigate the influence of folic acid supplementation on the follicular fluid concentrations of folate and total homocysteine and their relationship to the diameter of the follicle. Design: Observational study. Setting: Tertiary referral fertility clinic at the Erasmus MC, University Medical Center, Rotterdam, The Netherlands. Patient(s): Thirty-seven women undergoing IVF or intracytoplasmic sperm injection treatment. Intervention(s): No interventions other than routine stimulation treatment and the recommendation of folic acid supplementation. Main Outcome Measure(s): Concentrations of folate and total homocysteine in monofollicular and pooled follicular fluid and the diameter of the follicle. Result(s): Folic acid supplementation significantly increased folate and decreased total homocysteine concentrations in pooled follicular fluid. In monofollicular fluid, folate concentrations only were significantly increased in supplemented women. The total homocysteine concentration appeared to be significantly correlated with the diameter of the follicle (r = 0.27). Samples from single follicles were less prone to artifacts in the measurements of the folate and total homocysteine concentration. Conclusion(s): Preconception folic acid supplementation significantly alters both folate and total homocysteine concentrations in follicular fluid. The correlation between the diameter of the follicle and total homocysteine concentration in follicular fluid warrants further investigation.