With 32 lactating Holstein cows we evaluated physiological effects of gossypol and cyclopropenoid fatty acids in diets containing 18.5% whole cottonseed (dry matter) based on corn, corn silage, and soybean meal. All cows consumed a control diet for the first 2 wk of lactation and then were assigned to either control or whole cottonseed diet for the remainder of their lactation. Milk production, milk fat and protein percentages, and daily dry matter intake were measured. Dry matter intakes were less for cows consuming the whole cottonseed diet, but net energy intake was similar for all diets. Milk fat from cows consuming whole cottonseed contained detectable concentrations of cyclopropene fatty acids. Total lipid in plasma, total serum cholesterol, serum gossypol, and apparent liver gossypol concentrations were greater in cows fed whole cottonseed. Gossypol and cyclopropenoid fatty acids appeared to be absorbed from the gut of cows fed whole cottonseed. Small amounts of gossypol in serum and liver tissue and small amounts of cyclopropene fatty acids in adipose tissue lipids and milk fat indicate a need to elucidate the significance of these physiologically active compounds in the human diet and their biological effects on lactating dairy cows.