Objective: To investigate the relation between lactation and markers of folate and vitamin B12 (B12) deficiency in women with and without vitamin supplementation. Design: A 9-month follow-up study. Subjects and methods: Blood samples from 91 women, who gave birth to a single healthy child, were collected 3 weeks, 4 and 9 months postpartum and analysed for circulating level of homocysteine (tHcy), methylmalonic acid (MMA), folate and B12. The participants were categorized as exclusively, partly or not breast-feeding dependent on the degree of lactation 4 months postpartum. During follow-up, lifestyle factors were recorded by structured interviews. Results: Among 72 exclusively breast-feeding women, the median (10-90% percentile) tHcy was 5.8 (3.1-8.3) mmol/l 3 weeks postpartum, 6.1 (4.1-10.3) mmol/l 4 months postpartum and 5.3 (3.6-8.7) mmol/l 9 months postpartum. At 9 months postpartum, none of the women breast-fed exclusively. No significant change occurred in the concentration of B12 and folate. Exclusively breast-feeding women without vitamin supplementation had higher median tHcy than supplemented exclusively breast-feeding women 4 and 9 months postpartum (7.0 vs 5.4 mmol/l (Po0.001) and 5.8 vs 4.5 mmol/l (P ¼ 0.003), respectively). Six women had increased (415 mmol/l) tHcy; four of these were unsupplemented and exclusively breast-feeding. Conclusion: We found no overall indication of depletion of the folate and B12 stores during the lactation period in this population. However, folate-supplemented women had lower tHcy and higher folate levels, suggesting a beneficial effect of supplementation with folate throughout lactation. Sponsorship: The Biomedical Laboratory Scientist Education and Research Fund and LEO Pharma Research Foundation supported this study. DPC Scandinavia, Denmark donated reagents for the folate and tHcy analysis.