Four lactating Holstein cows producing about 21 kg milk/day were fed complete rations containing 0, 5, 15, and 25% whole cottonseed in a 4 X 4 Latin square design. Diets were fed ad libitum. Periods were 21 days in length with total collections of feces and urine during the last 7 days of each period. Digestibilities o nitrogen, lipid, and energy increased with increasing cottonseed in the diet. There were no significant effects of cottonseed on digestibility or availability of fiber components, calcium, phosphorous, and magnesium. Cottonseed feeding did not affect dry matter intake or milk yield but did increase yields of milk fat and fat-corrected milk and decrease milk protein and solids-not-fat percentages. Synthesis of fatty acid in the mammary gland was depressed by about 50% on the 25% cottonseed diet, but transfer of dietary fat resulted in twofold increases in yields of stearic and oleic acids. Hydrogenation of cottonseed fatty acid in the rumen resulted in a fourfold increase in yield of trans oleic acid in milk fat. Samples from bulk milk tank and data collected from 55 commercial dairies showed similar changes in fatty acid composition of milk from cows fed whole cottonseed. There was no apparent effect of feeding up to 2.9 kg cottonseed dry matter per cow per day on calving interval or on incidence of displaced abomasum, ketosis, milk, fever, or retained placenta.