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The objectives of this study were to determine the nutrient composition of grass-fed beef in the United States for inclusion in the USDA National Nutrient Database for Standard Reference, and to compare the fatty acid composition of grass-fed and conventionally fed (control) beef. Ground beef (GB) and strip steaks (SS) were collected on 3 separate occasions from 15 grass-fed beef producers that represented 13 different states, whereas control beef samples were collected from 3 regions (Ohio, South Dakota, and Texas) of the United States on 3 separate occasions. Concentrations of minerals, choline, vitamin B(12), and thiamine were determined for grass-fed beef samples. Grass-fed GB samples had less Mg, P, and K (P < 0.05), and more Na, Zn, and vitamin B(12) (P < 0.05) than SS samples. Fat color, marbling, and pH were assessed for grass-fed and control SS. Subjective evaluation of the SS indicated that grass-fed beef had fat that was more yellow in color than control beef. Percentages of total fat, total cholesterol, and fatty acids along with trans fatty acids and CLA were determined for grass-fed and control SS and GB. Grass-fed SS had less total fat than control SS (P = 0.001), but both grass-fed and control SS were considered lean, because their total fat content was 4.3% or less. For both GB and SS, grass-fed beef had significantly less (P = 0.001 and P = 0.023, respectively) content of MUFA and a greater content of SFA, n-3 fatty acids, CLA, and trans-vaccenic acid than did the control samples. Concentrations of PUFA, trans fatty acids, n-6 fatty acids, and cholesterol did not differ between grass-fed and control ground beef. Trans-vaccenic acid (trans-11 18:1) made up the greatest concentration of the total trans fats in grass-fed beef, whereas CLA accounted for approximately 15% of the total trans fats. Although the fatty acid composition of grass-fed and conventionally fed beef was different, conclusions on the possible effects of these differences on human health cannot be made without further investigation.