The objective of this study is to induce the primary characteristics of phenylketonuria in infant rats during the first 2 weeks after birth. The critical biochemical parameter in the development of phenylketonuria is the elevation of plasma phenylalanine while tyrosine is maintained at a relatively low level. A PKU-like condition was induced in infant rats during the first 2 weeks after birth using a modification of our previously published procedure for the development of a temporary (1 to 3 days) PKU-like condition. Phenylalanine was administered by stomach intubation every 6 hours (starting at 6:00 a.m.) at a dose level of 400mg per kg body weight (after birth-day 2 thru 5) and 500mg per kg body weight (day 6 thru 14). Amethopterin was given at 0.00625 or 0.0125mg per kg body weight (day 3 thru 14) and p-chlorophenylalanine at 50 mg per kg body weight (day 5 thru 14) at 9:00 a.m. and 9:00 p.m. At the times measured (6,10 and 14 days) plasma phenylalanine/tyrosine (P/T) ratios were elevated from a normal value of the or less to values ranging from 6 to 15. During the second week after birth a staggering gait, abnormal stance and decreased social behavior were also observed. None of these PKU-like characteristics were apparent in the three control groups receiving (a) no phenylalanine or inhibitors, (b) phenylalanine alone, or (c) inhibitors alone. The establishment of these primary biochemical characteristics of phenylketonuria by stomach intubation of phenylalanine and a combination of low dose levels of enzyme inhibitors to infant rats provides an experimental system which should he valuable for extensive biochemical, histological and behavioral studies in phenylketonuria.