Elevated plasma total homocysteine (tHcy) concentrations are associated with lower folate, vitamin B-12, and vitamin B-6 status and are considered an independent risk factor for cardiovascular disease in developed countries, but data in developing countries are limited. We conducted a cross-sectional study to explore tHcy status and its association with plasma B vitamin status in 2471 Chinese men and women aged 35 to 64 y, living in the urban and rural areas of the northern and the southern regions of China. Blood samples were also collected in 2 seasons (spring and fall). The geometric mean plasma tHcy concentration was significantly higher in the north (adjusted geometric mean, 95% CI; 13.0 μmol/L, 12.6-13.3) than in the south (9.1, 8.9-9.4) after controlling for gender, area (urban and rural), age, and season (spring and fall). Twenty-eight percent of northerners and 7% of southerners had plasma tHcy concentrations ≥16.0 μmol/L, a level used to define hyperhomocysteinemia. Within each region, men had higher plasma tHcy concentrations than women (16.1 vs. 10.6 μmol/L in the north, and 10.7 vs. 7.9 μmol/L in the south) and 40% of the northern men had hyperhomocysteinemia. Generally, individuals living in the urban areas had 30% (95% CI, 1.0-1.6) greater odds of having high tHcy levels (≥16 umol/L) than those living in the rural areas. Low plasma concentrations of folate, vitamins B-12 and B-6, older age, being male, and living in urban areas were all independently associated with elevated tHcy, with low folate as the strongest determinant. J. Nutr. 137: 407-413, 2007.