Maternal vitamin B-12 deficiency and hyperhomocysteinemia predict poor pregnancy outcome, foetal adiposity and insulin resistance. In India amongst practicing clinicians and policy makers there is little appreciation of widespread vitamin B-12 deficiency. We investigated 163 (86 rural, 77 urban) pregnant women attending antenatal clinics in a rural health centre and a referral hospital in the city of Pune, at 17, 28, and 34 weeks gestation for vitamin supplements, and circulating concentrations of vitamin B-12, folate, and total homocysteine. At enrolment 80% rural and 65% urban women had low vitamin B-12 but only two rural women had low folate concentrations. During pregnancy 85% rural and 95% of urban women received folic acid; 12% rural and 84% urban women also received vitamin B-12. In women receiving no supplementation (n=17) plasma vitamin B-12 and folate did not change from 17 to 34 weeks gestation, but homocysteine increased (p<0.05). Homocysteine concentrations at 34 weeks gestation in women receiving only folic acid (n=71, mean 8.4 (95% CI 7.8, 9.1) mu mol/L) were comparable to the unsupplemented group (9.7 (7.3, 12.7), p=0.15), but women who received a total dose of > 1000 mu g of vitamin B12 up to 34 weeks (n=42, all with folic acid) had lower concentrations (6.7 (6.0, 7.4), p<0.001). Increasing dose of vitamin B-12 (r(s)=-0.3 1, p=0.006) but not folic acid (r(s)=-0.19, p=0.11) was associated with lower plasma total homocysteine concentration. In vitamin B-12 insufficient, folate replete pregnant women, vitamin B-12 supplementation is associated with a reduction of plasma total homocysteine concentration in late pregnancy.