Forty-two lactating dairy cows were used to determine the interaction between folic acid and methionine dietary supplementation on protein metabolism at 6 and 25 weeks of lactation. Treatments were tested according to a 2 × 3 factorial arrangement, with two levels of methionine (0 vs 18 g of rumen protected methionine) and three levels of folic acid (0, 3, or 6 mg/d per kg of BW of pteroylmonoglutamic acid), equally distributed in 7 blocks of 6 cows each. Whole body leucine kinetics were determined using a constant infusion of L[1-¹³C]leucine (1.8 mmol/h). Neither milk production, protein yield or leucine kinetics were affected by treatments. Milk production (45.5 to 35.4 ± 0.85 kg/d) and protein yield (1.43 vs 1.22 ± 0.028 kg/d) were higher (both P<0.001) at 6 vs 25 weeks of lactation. However, total whole body leucine irreversible loss rate was not affected by stage of lactation, but fractional oxidation increased as lactation advanced (0.136 vs 0.156 ± 0.0065; P=0.03). Whole body protein synthesis was not affected by the stage of lactation (4.14 and 4.08 ± 0.091 kg/d), but the partition of this synthesis was altered, with 0.453 vs 0.403 ± 0.0095 (P<0.001) of leucine used for protein synthesis directed towards milk output. However, absolute rates of non-milk protein synthesis were not affected by the stage of lactation. Although concentrations of IGF-1, insulin and somatotropin varied with stage of lactation, they did not correlate with protein metabolism. In the dairy cow, the high demand for milk production still represents an important portion of the leucine used for protein synthesis until mid-late lactation.