Four multiparous lactating Holstein cows, fitted with cannulas in the rumen, duodenum, and ileum, were used in a trial with a 4 x 4 Latin square design to examine the effect of source and degradability of dietary CP supplements on AA flow and absorption in the small intestine. The CP supplements consisted of fish meal or meat meal as the animal by-products supplement and soybean meal or heated soybean meal as the plant by-products supplement. Fish meal and heated soybean meal constituted the high RUP diets, and meat meal and soybean meal constituted the low RUP diets. Dry matter intakes were similar among treatments. However, intakes of OM, rumen-degradable OM, total CP, and RDP were affected by the source of the CP supplement and were higher for diets with plant by-products than for those diets with animal by-products. Data were similar for the calculated in situ digestibilities and for true in vivo digestibilities of rumen-degradable OM. Efficiency of bacterial CP synthesis was similar among diets; however, nonbacterial CP flow to the duodenum and OM, CP, and disappearance of AA from the small intestine were affected by the interaction of supplement source and degradability. Heat treatment of soybean meal reduced the availability of most of the essential AA for absorption in the small intestine by 2.3% compared with that of soybean meal; the biggest such reduction (20%) occurred for Met. Availability of AA in the small intestine was affected by source and degradability of the CP dietary supplement. Heat treatment at 140 degrees C for 2.5 h reduced the availability of Met absorption from the small intestine. Although the differences among availabilities of the individual AA in the small intestine ranged from 80 to 84% among treatments, for high yielding cows, these differences may have practical significance.