Background: Prematurity may influence the levels of amino acids, enzymes, and endocrine markers obtained through newborn screening. Identifying which analytes are the most affected by degree of prematurity could provide insight into how prematurity impacts metabolism. Methods: Analytes from blood spots assayed by Newborn Screening Ontario between March 2006 and April 2009 were used in this analysis. We examined the associations between the degree of prematurity and the levels of amino acids, enzymes, and endocrine markers in all newborns with and without adjustment for birth weight, feeding status, sample timing, transfusion, and sex. results: Our analysis included the following cohorts: 373,819 children born at term (>36 wk gestation), 26,483 near-term children (33-36 wk gestation), 4,354 very premature children (28-32 wk gestation), and 1,146 extremely premature children (<28 wk gestation). Of the amino acids showing consistent trends across categories of prematurity, the levels of three amino acids (arginine, leucine, and valine) were at least 50% different between the cohorts of extremely premature and term children. The levels of 17-hydroxyprogesterone increased with increasing prematurity, while thyrotropin-stimulating hormone values consistently decreased with increasing prematurity. None of the three enzyme markers we examined showed a trend in levels across categories of prematurity. conclusion: This study demonstrates that children at different stages of prematurity are metabolically distinct. Future research should focus on the mechanism by which specific analytes are influenced by prematurity.i n population-wide newborn screening, dried blood spot samples are obtained usually within 24-72 h of birth from which levels of various analytes are measured (1,2). In Ontario and several other jurisdictions, these analytes include markers of amino acid metabolism, fatty acid oxidation, and endocrine function (3-7). Analyte results are used to calculate risk for rare inborn errors of metabolism and endocrine disorders.Children having positive screening results are sent for confirmatory testing.It is generally recognized that, aside from the presence of a rare metabolic or endocrine disease, several other factors can affect the analytes measured for newborn screening. One of these is gestational age (GA) at birth (8-11). Prematurity may influence analyte levels through a variety of mechanisms, including increased catabolic stress, lessened maturation of metabolic pathways and/ or organ systems, and differential distribution of other confounding variables such as feeding status (9,12). Identifying which analytes are the most affected by extent of prematurity could provide insight into how prematurity influences metabolism.In Ontario, Canada, virtually every child undergoes newborn screening (13). Results for all analytes measured are stored along with key demographic variables. We examined the associations between different degrees of prematurity and subclinical levels of specific analytes, after removing screen...