Background: It is not fully established whether the increasing risk of coronary artery disease (CAD) is associated with high plasma homocysteine levels or components of the homocysteine remethylation pathway, e.g. vitamin B<sub>12</sub> or 5-methyltetrahydrofolate (5-MTHF) in plasma and red blood cells (RBC). In this study, we tested the hypothesis that 5-MTHF in RBC, which represents the long-term folate status of individuals, may be a more reliable marker of homocysteine remethylation pathway disturbances, and its deficiency may be associated with CAD in Iranians. Methods: Plasma total homocysteine (tHcy), vitamin B<sub>12</sub>, and plasma and RBC 5-MTHF were measured in 200 angiographically documented patients and 200 controls matched for sex and age. Results: In the plasma, tHcy levels were significantly higher in cases compared to controls (geometric mean 12.9 ± 6.5 vs. 10.6 ± 5.6 µmol/l, p = 0.04). However, RBC 5-MTHF (527.2 ± 185.9 vs. 461.3 ± 117.9 nmol/l, p = 0.007) and vitamin B<sub>12</sub> (254.2 ± 132.8 vs. 182.2 ± 110.4 pmol/l, p = 0.04) were significantly higher in controls than patients. RBC 5-MTHF was a strong and independent predictor of plasma tHcy (β = –0.01, p = 0.003, r<sup>2</sup> = 0.19). Subjects in the lowest quartile of red-cell 5-MTHF had a 2.5-fold increased prevalence of CAD compared to subjects in the highest quartile. The association of CAD in the first quartile with red-cell 5-MTHF remained significant when adjusted for plasma tHcy, vitamin B<sub>12</sub>, hypertension and hypercholesterolemia (odds ratio, OR 2.3, confidence interval: 1.1–3.9, p = 0.01). However, the association between CAD in the highest quartile and plasma tHcy decreased and became insignificant when adjusted for red-cell 5-MTHF, vitamin B<sub>12</sub>, hypertension and hypercholesterolemia (OR 1.27, confidence interval: 0.96–1.69, p = 0.11). Conclusion: In this study, the association between CAD and low RBC 5-MTHF was stronger than with plasma 5-MTHF and plasma tHcy levels, indicating that RBC 5-MTHF may be a more stable parameter to study disturbances in the homocysteine remethylation pathway in Iranians.