OBJECTIVE: The purpose of this study was to investigate the long-term effect of hormone replacement therapy on total homocysteine and to study whether there was any difference in effect between opposed and unopposed hormone replacement therapy or whether the methylenetetrahydrofolate reductase C677T polymorphism was associated with the effect of hormone replacement therapy on total homocysteine. STUDY DESIGN: Two hundred nine healthy postmenopausal women were randomized to hormone replacement therapy (n = 103) or no substitution (n = 106) 5 to 7 years earlier. RESULTS: Women who received hormone replacement therapy had significantly lower total homocysteine concentrations than women in the control group; median total homocysteine values were 8.6 μmol/L and 9.7 μmol/L, respectively, in a per-protocol analysis (P = .02). The effect was comparable in all methylenetetrahydrofolate reductase genotypes, and no difference between unopposed and opposed hormone replacement therapy could be demonstrated. Similar results were obtained when an intention-to-treat analysis was performed. CONCLUSION: Long-term hormone replacement therapy results in lower total homocysteine concentrations in all methylenetetrahydrofolate reductase genotypes without demonstrable difference in effect between unopposed and opposed hormone replacement therapy.