To clarify the role of methylenetetrahydrofolate reductase (MTHFR) thermolability and vitamin status in the pathogenesis of hyper-homocysteinemia (HHC) we measured fasting homocysteine, plasma B 1 2 , and red blood cell (RBC) folate in 100 coronary artery disease (CAD) patients with normal lipids and in 27 age- and gender-matched angiographically normal controls. MTHFR status was determined in the CAD group. MTHFR thermolability was present in 17% of CAD subjects, and fasting HHC (>2 SD above the control mean) in 39%. Thirty-six percent of CAD subjects with HHC were MTHFR thermolabile, and had significantly higher homocysteine (27 μmol/L) than non-thermolabile hyperhomocysteinemic subjects (20 μmol/L, P < 0.05). Mean RBC folate and plasma B 1 2 were lower in the hyperhomocysteinemic MTHFR thermolabile subjects than in the MTHFR thermolabile subjects with normal homocysteine levels (P < 0.05, P = 0.38 respectively). In the non-thermolabile hyperhomo-cysteinemic subjects, mean plasma B 1 2 , but not RBC folate, was lower than in the non-thermolabile normal homocysteine subjects (P < 0.001). By correcting first for folate and then for vitamin B 1 2 , strong and equal inverse relationships between homocysteine and folate and between homocysteine and plasma B 1 2 were demonstrated. The majority (74%) of all hyperhomocysteinemic subjects had plasma B 1 2 levels in the lower half of the accepted normal range for plasma B 1 2 . The majority (71%) of MTHFR thermolabile subjects were also clustered in the lower half of the normal range for RBC folate. Furthermore, thermolabile subjects had higher homocysteine than non-thermolabile subjects at similar vitamin B 1 2 folate levels. In conclusion, our data suggests that current normative ranges for vitamin B 1 2 and folate are too low. Relative B 1 2 deficiency leading to HHC is common in our CAD populations with normal lipids and account for the majority of hyperhomocysteinemia. MTHFR thermolability confers increased sensitivity to relative deficiencies of both plasma B 1 2 and folate, resulting in markedly elevated homocysteine.