Cardiovascular disease remains a major health problem in the United States. Consumption of soluble fiber, like that found in oats, has been recognized as beneficial in decreasing blood cholesterol levels and other cardiovascular risk factors. Barley has high amounts of soluble fiber but is not extensively consumed in the U.S. diet. This study investigated whether consumption of barley reduces cardiovascular risk factors comparably to reductions observed with other soluble fiber sources. Moderately hypercholesterolemic subjects consumed controlled American Heart Association Step 1 diets for 17 weeks. After a 2-week adaptation period, whole-grain foods containing 0, 3, or 6 g of soluble fiber (beta-glucan) from barley per day were included in the Step 1 menus. Diets were consumed for 5 weeks each and fed in a Latin-square design. Total and LDL cholesterol were significantly lower when the diet contained 3 or 6 g of beta-glucan from barley; the greatest change occurred in men and postmenopausal women. HDL cholesterol and triacylglycerol concentrations did not differ among the three levels of soluble fiber. Large LDL particle size significantly decreased when whole grains were incorporated into the three diets. Postmenopausal women had significantly larger LDL particle size. These results indicate that barley may be an effective addition to a healthy diet to lower total and LDL cholesterol in both men and women.