In vivo and in situ trials were conducted to investigate the utilization of alfalfa protein concentrate as a protein supplement for lactating dairy cows. In the in vivo trial, four Holstein cows fitted with ruminal, duodenal, and ileal cannulae were used in a crossover experiment to measure site and extent of nutrient digestion. Isonitrogenous and isoenergetic diets containing alfalfa protein concentrate or soybean meal were fed four times daily. Sixty percent of dietary total N was provided by the test proteins. Ruminal ammonia and total VFA concentrations were lower in cows fed alfalfa protein concentrate. Fractional digestion coefficient of organic matter (52 vs. 43%) and degradation of dietary protein (57 vs. 47%) in the rumen were slightly lower in cows fed alfalfa protein concentrate. The ratio of amino acid flow to the small intestine to dietary intake of amino acid was higher in cows fed alfalfa protein concentrate. In the in situ trial, rate of N disappearance (2.4 vs. 9.5%.h-1) and potential protein degradability (35 vs. 68%) were lower for alfalfa protein concentrate. Results suggest that alfalfa protein concentrate may be more resistant to ruminal degradation than soybean meal and could be utilized as a relatively undegradable protein source for milk production.