This study evaluated the effect of varying levels of tyrosine intake on the estimation of phenylalanine hydroxylation. Healthy men were fed 1 g protein kg(-1) x d(-1) for a 2-day period. On the third day, subjects consumed a formula diet containing 1 g protein kg(-1) x d(-1) hourly over 10 hours, and primed hourly oral doses of L-[15N]phenylalanine and L-[3,3-2H2]tyrosine for the last 6 hours. Each subject was studied at 7 levels of tyrosine intake (3.0, 4.5, 6.0, 7.5, 9.0, 10.5, and 12.0 mg x kg(-1) x d(-1)) at a constant intake of phenylalanine (9 mg x kg(-1) x d(-1), 4.55 micromol x kg(-1) x h(-1)). Phenylalanine hydroxylation was estimated from the ratio of plasma amino acid isotope enrichment of [15N]phenylalanine and [15N]tyrosine and the tyrosine flux estimated from [2H2]tyrosine enrichment. Phenylalanine and tyrosine fluxes showed no significant response to alterations in the intake of tyrosine. Linear regression analysis showed a significant response such that the rate of phenylalanine hydroxylation decreased as tyrosine intake increased (R2 = .21; P = .003). The mean rates of phenylalanine hydroxylation were 3.89 to 8.06 micromol x kg(-1) x h(-1). Given model uncertainties, the apparent protein breakdown observed at tyrosine intake levels less than 10.5 mg x kg(-1) x d(-1), and the significant differences observed between the present data and our prior data, we cannot estimate the tyrosine requirement with any degree of certainty with the present hydroxylation results.