Four lactating Holstein cows fitted with rumen cannulae and T-type cannulae in proximal duodenum and terminal ileum were used to measure protein degradation in the rumen and amino acid flow and absorption in the small intestine. Soybean meal, whole soybeans, and whole soybeans extruded at 132 and 149°C provided 50% of the protein in diets that contained 51% grain, 36% corn silage, and 13% alfalfa hay (dry matter). Spot samples of digesta were collected from duodenum and ileum during 96 h, and lanthanum was an indigestible marker to estimate flow and digestibility of nutrients. With diaminopimelic acid as a microbial marker, apparent degradations of dietary crude protein in the rumen were 73, 80, 66, and 60% for diets containing soybean meal, whole soybeans, and whole soybeans extruded at 132 and 149°C. Because of the extensive degradation of protein with the raw soybean diet, less total amino acids reached the duodenum, and because of decrease of availability, absorption from the small intestine (g/day) was lowest with this diet. Feeding diets containing extruded whole soybeans increased availability of total essential amino acids in the small intestine compared with diets containing soybean meal and whole soybeans. Absorption from the small intestine (g/day and percent entering) of individual amino acids was generally higher for extruded whole soybean diets.