The methionine cycle and methyl group metabolism are implicated in the long-term programming of metabolism. Diets deficient in folic acid, methionine and choline have been fed to pregnant rats to examine the effects on amino acid metabolism, choline reserves and DNA methylation in dam and fetuses. Animals were fed folate-deficient, folate-deficient with low methionine, folate-deficient with low choline and folate-deficient, low-methionine, low-choline diets starting 2 weeks before mating. The dams and their fetuses were subsequently killed on day 21 of gestation for analysis. Diets low in methionine reduced fetal and maternal weight. Folate deficiency increased the concentrations of homocysteine, glycine, serine and threonine in the maternal plasma, and this was exacerbated by the low-methionine diets. The changes in the amino acid profile in the fetal serum were similar but less pronounced. This result suggests that fetal metabolism was less perturbed. Folate deficiency increased free choline in the maternal liver at the expense of phosphocholine stores. It has been suggested that a deficiency in methyl donors in the diet during pregnancy may impact on key methylation reactions, including the methylation of DNA. Despite widespread changes in the metabolism of choline and amino acids, there was no change in the global methylation of cytosine in DNA from either maternal or fetal livers. This suggests a more indirect mechanism in which gene-nutrient interactions modify the process of differential methylation during development.