The long-chain polyunsaturated fatty acids are considered to be of major health importance, and recent studies indicate that their endogenous metabolism is influenced by B-vitamin status and smoking habits. We investigated the associations of circulating B-vitamins and smoking habits with serum polyunsaturated fatty acids among 1,366 patients who underwent coronary angiography due to suspected coronary heart disease at Haukeland University Hospital, Norway. Of these, 52% provided information on dietary habits by a food frequency questionnaire. Associations were assessed using partial correlation (Spearman's rho). In the total population, the concentrations of most circulating B-vitamins were positively associated with serum n-3 polyunsaturated fatty acids, but negatively with serum n-6 polyunsaturated fatty acids. However, the associations between B-vitamins and polyunsaturated fatty acids tended to be weaker in smokers. This could not be solely explained by differences in dietary intake. Furthermore, plasma cotinine, a marker of recent nicotine exposure, showed a negative relationship with serum n-3 polyunsaturated fatty acids, but a positive relationship with serum n-6 polyunsaturated fatty acids. In conclusion, circulating B-vitamins are, in contrast to plasma cotinine, generally positively associated with serum n-3 polyunsaturated fatty acids and negatively with serum n-6 polyunsaturated fatty acids in patients with suspected coronary heart disease. Further studies should investigate whether B-vitamin status and smoking habits may modify the clinical effects of polyunsaturated fatty acid intake.