Effects of feeding varying amounts of total gossypol from whole cottonseed and cottonseed meal were evaluated in 30 lactating Holstein cows. After a 14-d pretreatment period, cows were assigned for 42 d to one of five treatments: control (diet A); 1040 mg/kg of total gossypol, and 989 mg/kg of free gossypol from whole cottonseed (diet B); 900 mg/kg of total gossypol and 64 mg/kg of free gossypol from cottonseed meal (diet C); 960 mg/kg of total gossypol and 531 mg/kg of free gossypol with equal amounts of total gossypol from whole cottonseed and cottonseed meal (diet D); or 1922 mg/kg of total gossypol and 1050 mg/kg of free gossypol with equal amounts of total gossypol from whole cottonseed and cottonseed meal (diet E). Concentrations of plasma gossypol and its isomers were directly proportional to free gossypol intake. Plasma gossypol concentrations plateaued after 35 d on treatment, and they were highest in cows receiving diet E. At 42 d on treatment, erythrocyte fragility was higher in the cows receiving the diet E, but it did not differ among other treatments. Dry matter intakes were similar for all groups. Cows receiving diet E produced more milk and 3.5% fat-corrected milk, but milk protein content decreased. Feeding diets containing 1900 mg/kg of total gossypol and 1050 mg/kg of free gossypol for 42 d resulted in increased plasma gossypol concentrations and erythrocyte fragility, but no detrimental impact on lactation performance were observed.