Smoking is associated with increased plasma homocysteine levels, and both are associated with an increased risk of cardiovascular disease. However, little information is available on the effects of passive smoking on the level of homocysteine in nonsmokers. We analyzed the data of self-reported never-smokers (aged > or = 20 years, n = 3,232), who were from the Third National Health and Nutrition Examination Survey. We quantified the passive nicotine exposure by dividing the never-smokers into quartiles as based on the serum cotinine values. Multiple linear and logistic regression models were used to determine any independent relationships between serum cotinine concentration and levels of homocysteine, vitamin B12, and folate. An elevated homocysteine level was defined as a concentration greater than the 80th percentile. A reduced folate or vitamin B12 level was defined as a concentration less than the 20th percentile.After adjusting for age, gender, body mass index, race, folate and vitamin B12 levels, increased cotinine levels (quartile III and IV) were found to be associated with hyperhomocysteinemia. There was a strong nonlinear increase in the serum homocysteine levels across the quartiles of cotinine. Multivariate analysis showed that age, male gender, non-Caucasian, low levels of folate and vitamin B12, and increased serum cotinine (quartile II-IV) were independently associated with elevated homocysteine levels. In conclusion, these findings indicate that passive smoke exposure in never-smokers is positively and independently associated with plasma homocysteine levels in a dose-dependent manner. These findings may help further determine the link between passive smoking and cardiovascular events.