There is evidence that ruminant trans fatty acids (FA), such as vaccenic acid (VA), do not increase risk of coronary heart disease, however this is poorly investigated. The objective was to investigate the effect of butter with a naturally high content of VA, and concomitant higher content of MUFA, on risk markers of coronary heart disease. 42 healthy men were given 115 g butter/d high in VA (V), or low in VA (C) for 5 weeks in a double-blinded, randomized, parallel intervention study. Compared to C, intake of V resulted in a 6% lower total cholesterol (P=0.05) and a 9% lower HDL cholesterol concentration (P=0.002), whereas the ratio of total cholesterol to HDL cholesterol did not differ between the groups. FA composition of lipid classes reflected the FA proportion of the test butter. No other differences were observed. Conclusion, butter high in ruminant trans FA decreased in total cholesterol and HDL cholesterol compared to control butter with higher amounts of saturated fat. We suggest that the differences were due to the greater content of MUFA and a lesser content of SFA of the butter rich in ruminant trans FA, rather than the content of VA per se. Supp. by Danish Dairy Res. Found. and The Danish Res. Development Program for Food Techn (Foetek).