Common chronic diseases including cardiovascular disease (CVD) are the leading cause of morbidity and mortality in developed countries. These diseases are multifactorial in origin with both genetic and environmental components that act through a complex network of gene-gene and gene-environment interactions to generate the health or disease phenotype. The folate metabolic network plays an important role in a variety of fundamental intracellular functions including remethylation of homocysteine to methionine, DNA synthesis and repair, DNA methylation, protein synthesis and cell-signaling. The overall objective of this work is to understand the role of genetic variation in the human folate metabolic network in cardiovascular risk. We have focused on five potentially-functional candidate SNPs in four genes involved in sequential reactions, namely methylene-tetrahydrofolate reductase (MTHFR), methylene-tetrahydrofolate dehydrogenase (MTHFD), methionine synthase (MTR), and cystoplasmic serine hydroxymethyl transferase (cSHMT). We use data from nested case control studies of cardiovascular disease conducted in the framework of two large epidemiological cohort studies, the Nurses? Health Study (NHS) and the Normative Aging Study (NAS). Our main findings are the presence of gene-nutrient interaction between folate and the MTHFR 677 polymorphism in predicting serum homocysteine levels, the presence of gene-gene interaction between the MTHFR 677 and MTHFD 1958 polymorphisms in predicting CVD risk, the presence of gene-gene interaction between the MTHFR 1298 and MTR 2756 polymorphisms, strengthening of the above two interactions with inclusion of serum homocysteine levels in the models, and the partial replication in a nested case-control study of women of a gene-gene interaction between MTHFR 677 and cSHMT 1420 polymorphism previously detected in a study on men. In summary, we have found evidence for the presence of gene-gene interaction between variants in genes encoding sequential reactions in the folate metabolic network. Lack of mediation by homocysteine suggests that other folate-related markers need to be studied to understand the pathophysiologic route from genotype to disease phenotype. Our findings suggest the importance of evaluating gene-gene interactions, especially among genes with functional connections, in epidemiologic studies of complex disease in general, and cardiovascular disease in particular.