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Whole tomato seeds, a novel by-product feedstuff, were fed to lactating Holstein cows to determine the nutritive value of whole tomato seeds by replacing whole cottonseed in the total mixed ration. Four primiparous and 4 multiparous Holstein cows were used in a 4×4 Latin square design and fed 1 of 4 total mixed rations. Whole tomato seeds replaced whole cottonseed on a weight-to-weight basis for lipid. The proportion of whole tomato seeds to whole cottonseed in the diets were 100:0, 50:50, 25:75, and 0:100 on a lipid basis. Thus, tomato seeds were 4.0, 2.4, 1.1, and 0% of the ration dry matter, respectively. Milk yield and the concentrations and yields of protein, lactose, and solids-not-fat did not differ for the effect of diet. However, milk fat concentration decreased and milk fat yield tended to decrease as whole tomato seeds replaced whole cottonseed. Intakes of dry matter, lipid, and crude protein did not differ. Whole-tract apparent digestibility of dry matter and ash-free neutral detergent fiber did not differ, but digestibility of total fatty acids and crude protein decreased with increasing proportion of whole tomato seeds. Urea concentration in milk and plasma both decreased with increasing whole tomato seeds. Fecal concentration of linoleic and α-linolenic acids increased with increasing whole tomato seeds, suggesting that seeds were passing out of the digestive tract undigested. The concentrations of C18:2n-6 and C18:3n-3 in milk fat had small increases, but their yields were not different, suggesting that only a small amount of whole-tomato-seed lipid might have been digested postruminally. Amounts of trans C18:1 fatty acids in milk fat were higher with increasing whole cottonseed, which might suggest a shift in rumen biohydrogenation pathways. At the level of feeding used in the current study, whole tomato seeds replaced whole cottonseed in the diet of lactating dairy cows without a change in production.