The objective of this study was conducted to evaluate the effects of replacing alfalfa hay with rice straw or corn stover on amino acid (AA) profiles of ruminal microbes, rumen undegradable protein (RUP), and gastrointestinal contents in lactating cows. Eighteen Holstein dairy cows were individually fed, and randomly assigned into one of 3 treatments. Isonitrogenous diets contained similar concentrate and 15% corn silage, with 3 forage sources (DM basis): 23% alfalfa hay and 7% Chinese wild rye hay (AH); 30% corn stover (CS); and 30% rice straw (RS). After 14-wk feeding, all the cows were slaughtered to collect the samples of rumen fluid and gastrointestinal contents. The AA were analyzed with an AA analyzer (Model L-8800, Hitachi, Tokyo, Japan). The variance of the data was analyzed as a completely randomized design using PROC MIXED of SAS. The AA profiles of ruminal microbes were similar among 3 treatments except for the lower value of Asp in diet RS than in CS (P < 0.01) or AH (P < 0.02) and the greater value of Gly in AH than in RS (P = 0.02). Significant differences were found in the AA profiles among ruminal microbes, fluid, and digesta, with greater percentage of essential AA in digesta than in microbes or fluid (P < 0.01), and in microbes than in rumen fluid (P < 0.01). The free AA concentration in jejunum was the greatest, but the peptide-binding AA concentration was the greatest in duodenum, resulting in lower ratio of peptide-binding AA to free AA. In addition, the free essential AA profiles in jejunum fluids for all the 3 diets were similar with the recommended digestible AA and milk AA, with the ratio of Lys to Met at 3:1. Most of AA of RUP was independent on forage sources except for Phe (P = 0.035) and Gly (P = 0.05). There was linear correlation between dietary AA profile and AA composition in ruminal fluids (P < 0.01). The AA profiles of ruminal microbes and RUP were constant and independent on forage sources. It is indicated that the AA profile of metabolizable protein may be constant independent on dietary effects.