Lysine requirement was determined in seven adult males by examining the effect of varying dietary lysine intake on phenylalanine flux and oxidation under dietary conditions of adequate energy and phenylalanine (14 mg.kg-1 x day-1) and excess tyrosine (40 mg.kg-1 x day-1). Phenylalanine flux was determined from primed, constant intravenous infusions of L-[1-13C]phenylalanine (1.2 mg.kg-1 x day-1) and L-[ring-2H5]phenylalanine (0.5 mg.kg-1 x day-1) and measurement of isotopic enrichments of phenylalanine in plasma. Phenylalanine flux was not affected by graded increases in dietary lysine intake or by the isotope infused. Mean phenylalanine conversion to tyrosine was low (3.4%) and not significantly affected by lysine intake. Phenylalanine oxidation, estimated from the rate of 13CO2 released in expired air during the infusion of L-[1-13C]phenylalanine, decreased linearly as lysine intake increased to a break point that was interpreted as the mean dietary lysine requirement (37 mg.kg-1 x day-1). At lysine intakes of > 37 mg.kg-1 x day-1 phenylalanine oxidation was low and constant. Plasma lysine concentrations supported this estimate of requirement. These data show that: 1) indicator amino acid oxidation can be used as a new method to determine amino acid requirements of humans and 2) the lysine requirement of adult males is three times greater than the World Health Organization recommendation of 12 mg.kg-1 x day-1.