Supplementation of rumen-protected (RP) AA is a practical approach to balance dietary Met and Lys for optimal milk production. Histidine may become limiting in low-CP dairy diets. However, dairy cows may tap on endogenous pools of His (e.g., anserine, carnosine) to meet requirements. We aimed to examine the effect of incremental amounts of RP-His on endogenous pools of His and AA metabolism in dairy cows. Eight multiparous Holsteins (130 ± 30 DIM) were used in a replicated 4 × 4 Latin squares design with 28-d experimental periods. Treatments included a basal diet composed (DM basis) of 50% corn silage, 15% haylage, and 35% concentrate supplemented with 0, 82, 164, and 246 g/d RP-His (Ajinomoto Inc.) and 11 g/d RP-Met (Smartamine M). Dietary His represented 2.06, 2.28, 2.52, and 2.75% of MP supply, respectively. The basal diet averaged 15.1% CP and 34.8% NDF. Blood (weekly) and muscle (biweekly) samples were collected and quantified for AA. Data were analyzed individually by week using the MIXED procedure of SAS, evaluating treatment effects with linear and quadratic contrasts. Plasma His increased linearly (P < 0.001) with RP-His from wk 1 (30.3 to 57.2 µM) to wk 4 (33.2 to 63.1 µM). Plasma Arg, Leu, Lys, and Phe decreased linearly (P = 0.05) and plasma Ile showed a trend for a linear decrease in response to RP-His in wk 3. In contrast, treatments did not result in linear responses for all other EAA across the 4 wk. Plasma carnosine increased linearly (P = 0.03) with RP-His supplementation in wk 1, 3, and 4, but not in wk 2. No linear effect was observed for plasma 3-Methyl-His. Inclusion of RP-His showed a linear effect on muscle His in both wk 2 (20.1 to 32.5 µM; P < 0.001) and 4 (20.3 to 35.5 µM; P < 0.001). Muscle Lys increased linearly in wk 2 (22.1 to 36.2 µM; P = 0.04), and muscle Thr tended to increase linearly in response to RP-His in wk 4. Muscle carnosine and anserine were not affected by treatments, but they showed quadratic trends in wk 2. Overall, RP-His was effective to increase His in plasma and muscle independent of the endogenous pools of His.