Twenty-four multiparous Holstein cows were randomly assigned at calving to treatment diets using a modified split-plot design to determine the effects of protein source on milk production and composition. The treatment diets consisted of an 80:20 combination of corn and alfalfa silages and whole cottonseed at 12% of the dietary dry matter (DM). The treatment diets were formulated to contain 17% crude protein (CP) and 20% acid detergent fiber on a DM basis. One of the following sources of supplemental CP was included in each treatment diet: 1) cottonseed meal, 2) cottonseed meal plus a rumen-undegradable protein (RUP) supplement, 3) soybean meal, and 4) soybean meal plus an RUP supplement. Cows were fed the initial treatment diet for 6 wk and then were switched to the other oilseed meal source but continued to receive the same amount of RUP during the second period of the study. Milk production and composition were not affected by treatment diet. Cows fed treatment diets without RUP supplementation consumed more DM and thus more CP. Supplementation with RUP resulted in greater milk production efficiency per unit of DM consumed. Cows fed treatment diets containing cottonseed meal had higher plasma gossypol concentrations than did cows fed treatment diets containing soybean meal. Plasma gossypol concentrations for all cows in each group were below the recommended upper limit that is considered to be safe. Data suggest that cottonseed meal in the diet can be substituted for soybean meal, resulting in similar milk production and composition.