Rumen-protected forms of Met contain an equimolar mixture of the D- and L-isomers. Only L-Met can be directly used for protein synthesis, but it is unclear how much of the D-isomer can be transformed into L-Met in ruminants. Four lactating dairy cows, with an average milk yield of 32.4 kg/d, received a basal diet (12.5% crude protein, supplying 1,718 g/d of metabolizable protein) in 12 equal meals per day plus an abomasal infusion of amino acids (590 g/d, casein profile without Met). They were used in 3 consecutive studies to determine utilization of D-Met. First, the cows each received portal vein infusions for 2 d of 5, 10, or 15 g/d of DL-Met in a Youden square. On the last day of each period, 6 arterial samples were collected at 45-min intervals. Concentrations of l-and D-Met were determined on a chiral column by gas chromatography-mass spectrometry. Portal infusion of 5, 10, and 15 g/d of DL-Met increased plasma total Met concentrations (19.7, 25.0, and 34.4 +/- 0.6 mu M) and the proportion of Met as d (19.4, 30.5, and 37.3 +/- 0.7%). The fractional removal of D-Met was 6 to 7 times lower than the fractional removal of L-Met, with mean half-lives of 52 versus 8 min, respectively. Second, the same cows were infused for 8 h with L[methyl-(2)H(3)] Met at 1.3 mmol/h; at 2 h, cows received a bolus injection i.v. of D-[1-(13)C] Met (6.8 mmol), and arterial samples were collected after 10, 20, 30, 40, 60, 90, 120, 150, 180, 240, 300, 360, 420, and 480 min. Expressed relative to L-[(12)C] Met; that is, as tracer: tracee ratios, enrichments of plasma D-[1-(13)C] Met and L-[1-(13)C] Met averaged 1.77 +/- 0.14 and 0.144 +/- 0.026, respectively, 10 min after the bolus injection and declined exponentially thereafter. A minimum of 75 +/- 3% of the D-[1-(13)C] Met was transformed into L-[1-(13)C] Met. Third, the cows received, in a crossover design, an abomasal infusion for 5 d of either DL-Met or L-Met (15 g/d) and, on the last day of each experimental period, blood samples were collected simultaneously from arterial, portal, hepatic, and mammary vessels. Arterial total Met concentrations were higher with DL-versus L-Met infusions (37.4 vs. 25.4 +/- 0.5 mu M), with 37.1 +/- 5.0% as D-Met. The mammary gland did not extract any D-Met. Hepatic removal of D-Met was observed, but was numerically lower than the fractional extraction of L-Met. In conclusion, much of the D-Met is transformed into L-Met by the dairy cow but at a slow rate. No uptake of D-Met occurs across the mammary gland but L-Met synthesized from the D-isomer elsewhere in the body can be utilized for milk protein synthesis.