An isotope dilution method using [1-13C]leucine (Leu) infusion together with open-circuit calorimetry was applied to determine the effect of metabolizable protein (MP) intake on rates of plasma Leu turnover and whole body protein synthesis (WBPS) in six heifers. WBPS rate was estimated from rate of plasma Leu turnover and Leu oxidation to carbon dioxide. The experiment consisted of three levels of MP intake and was conducted in a two 3×3 Latin square designs of three 21-day periods. The experimental diet consisted of mixed hay, maize and soybean meal. Dietary MP intake of each dietary treatment was 4·3, 4·5 and 4·9 g/kg BW0·75/day by changing maize and soybean meal weights. Metabolizable energy (ME) intake was similar for all dietary treatments. When plasma α-[1-13C]keto-isocaproic acid enrichments were used as markers indicating intracellular Leu enrichments, plasma Leu turnover rate (LeuTR) increased (P=0·012) and WBPS tended to increase (P=0·091) as MP intake increased. In contrast, plasma LeuTR and WBPS were not influenced if plasma [1-13C]Leu was taken to indicate intracellular Leu enrichments. Total and plasma Leu oxidation rates did not change but intracellular Leu oxidation increased (P=0·044) with increasing MP intake. In heifers, it is suggested that rates of plasma Leu turnover and WBPS are influenced by dietary MP intake, independent of ME intake, although the change in MP intake was relatively small.